Involvement of protein kinase C in agonist-stimulated goldfish ovulation

The effects of two protein kinase C (PKC) inhibitors, calphostin C and staurosporine, on the in vitro ovulation of goldfish (Carassius auratus) oocytes were investigated. Ovulation was stimulated by prostaglandin (PG) F2 alpha (PGF2 alpha, 2.0 micrograms/ml), by sodium orthovanadate (0.1 mM), by a combination of the phorbol ester phorbol 12-myristate-13-acetate (PMA, 0.1 micrograms/ml) and calcium ionophore A23187 (0.05 micrograms/ml), by thapsigargin (0.2 micrograms/ml), and by elevated pH (8.1). In addition, the effects of these inhibitors on the PKC activity of the goldfish follicle wall was determined by use of a specific peptide substrate phosphorylation assay. At 0.1 microM, staurosporine significantly blocked ovulation induced by all agents. However, at lower (0.01 microM) levels it blocked only PMA/A23187-induced ovulation. In contrast, calphostin significantly blocked only PMA/A23187-induced ovulation, although there was a decrease in pH-induced ovulation at lower calphostin concentrations. Both calphostin and staurosporine blocked follicular PKC activity at levels that were inhibitory to ovulation. In addition, staurosporine significantly blocked PKC activity at levels even lower than those needed to block ovulation. The combined results suggest that orthovanadate, PGF2 alpha, and thapsigargin do not require PKC activation for the induction of ovulation, whereas PMA/A23187 does.     

Characterization and assessment of a novel poly(ethylene oxide)/polyurethane composite hydrogel (Aquavene) as a ureteral stent biomaterial

Although endothelium-derived hyperpolarizing factor (EDHF) activity has been demonstrated in arteries from various species, EDHF has not been chemically identified, nor its mechanism of action characterized. To elucidate this mechanism, we tested the effect of EDHF on large-conductance Ca2+-activated K+ (K(Ca)) channels in porcine coronary artery smooth muscle cells. By using a patch-clamp technique, single-channel currents were recorded in cultured smooth muscle cells; the organ bath also contained a strip of porcine coronary with endothelium, which served as the source of endothelium-derived relaxing factor(s) including EDHF. Exposure of endothelium to 10(-6) M bradykinin activated K(Ca) channels in cultured smooth muscle cells in cell-attached patches. When the experiment was performed in the presence of 10 microM indomethacin and 30 microM N(G)-nitro-L-arginine (L-NNA), which block the generation of prostaglandin I2 (PGI2) and NO, respectively, K(Ca) channel activity was stimulated by bradykinin, indicating the direct involvement of EDHF in K(Ca) channel stimulation. Neither 10 microM methylene blue nor 25 microM Rp-cAMPS inhibited bradykinin-induced K(Ca) channel activity. In inside-out patches, the addition of bradykinin to the solution was without effect on K(Ca) channel activation. However, in the presence of 0.5 mM guanosine triphosphate (GTP) and 1.0 mM adenosine triphosphate (ATP) in the bath solution, K(Ca) channels was activated by bradykinin. In outside-out patches, the addition of bradykinin also increased K(Ca) channel activity, when GTP and ATP were added to the pipette solution. The addition of GDP-beta-S (100 microM) in the cytosolic solution completely blocked the activation K(Ca) channels induced by bradykinin in inside-out and outside-out patches. Pretreatment with 30 microM quinacrine, a phospholipase A2 inhibitor, or 3 microM 17-octadecynoic acid (17-ODYA), a cytochrome P450 inhibitor, in addition to indomethacin and L-NNA, abolished bradykinin-stimulated K(Ca) channel activity in cell-attached patches. Both 14,15-epoxyeicosatrienoic acid (EET) and 11,12-EET increased the open probabilities of K(Ca) channels in cell-attached patches. These results suggest that EDHF, released from endothelial cells in response to bradykinin, hyperpolarizes smooth muscle cells by opening K(Ca) channels. Furthermore, our data suggest that EDHF is an endothelium-derived cytochrome P450 metabolite of arachidonic acid. The effect of EDHF on K(Ca) channels is not associated with an increase of cAMP and cGMP. The activation of K(Ca) channels appears to be due to the activation of GTP-binding protein.     

3,4 dichlorobenzamil-sensitive, monovalent cation channel induced by palytoxin in cultured aortic myocytes

1. Smooth muscle cells were dispersed from rat aorta and then cultured. The action of palytoxin on rat aortic myocytes was analysed by measurement of 22Na+ uptake and single channel recording techniques. 2. Palytoxin induced an increase in 22Na+ uptake, with a concentration of 50 nM producing half-maximal activation. The action of palytoxin was inhibited by amiloride derivatives and by ouabain. The concentrations of inhibitor producing half-maximal inhibition were 10 microM for 3,4 dichlorobenzamil, 30 microM for benzamil, 100 microM for phenamil and 1 mM for ouabain. 3. In outside-out patches, palytoxin induced single channel currents that reversed near 0 mV with NaCl or KCl in the extracellular solution, but were outward with N-methyl-D-glucamine chloride or CaCl2 (110 mM), indicating that palytoxin induced a cation channel permeable to Na+ and K+ (PK/PNa = 1.2) but not to Ca2+ (PK/PCa > 30) or to N-methyl-D-glucamine (NMDG) (PK/PNMDG > 11) The unit channel conductance was 11-14 pS. 4. A high (> 0.1 mM) extracellular concentration of Ca2+ was necessary to observe channel activation by palytoxin. A high (150 mM) extracellular concentration of K+ partially prevented and reversed channel activation by palytoxin. 5. The channel activity was fully blocked by 3,4 dichlorobenzamil (20 microM) and partially blocked by phenamil (50 microM). It was not reduced by ouabain (200 microM).     

Pharmacological comparison of UTP- and thapsigargin-induced arachidonic acid release in mouse RAW 264.7 macrophages

1. Although stimulation of mouse RAW 264.7 macrophages by UTP elicits a rapid increase in intracellular free Ca2+ ([Ca2+]i), phosphoinositide (PI) turnover, and arachidonic acid (AA) release, the causal relationship between these signalling pathways is still unclear. In the present study, we investigated the involvement of phosphoinositide-dependent phospholipase C (PI-PLC) activation, Ca2+ increase and protein kinase activation in UTP-induced AA release. The effects of stimulating RAW 264.7 cells with thapsigargin, which cannot activate the inositol phosphate (IP) cascade, but results in the release of sequestered Ca2+ and an influx of extracellular Ca2+, was compared with the effects of UTP stimulation to elucidate the multiple regulatory pathways for cPLA2 activation. 2. In RAW 264.7 cells UTP (100 microM) and thapsigargin (1 microM) caused 2 and 1.2 fold increases, respectively, in [3H]-AA release. The release of [3H]-AA following treatment with UTP and thapsigargin were non-additive, totally abolished in the Ca2+-free buffer, BAPTA (30 microM)-containing buffer or in the presence of the cPLA2 inhibitor MAFP (50 microM), and inhibited by pretreatment of cells with pertussis toxin (100 ng ml(-1)) or 4-bromophenacyl bromide (100 microM). By contrast, aristolochic acid (an inhibitor of sPLA2) had no effect on UTP and thapsigargin responses. 3. U73122 (10 microM) and neomycin (3 mM), inhibitors of PI-PLC, inhibited UTP-induced IP formation (88% and 83% inhibition, respectively) and AA release (76% and 58%, respectively), accompanied by a decrease in the [Ca2+]i rise. 4. Wortmannin attenuated the IP response of UTP in a concentration-dependent manner (over the range 10 nM-3 microM), and reduced the UTP-induced AA release in parallel. RHC 80267 (30 microM), a specific diacylglycerol lipase inhibitor, had no effect on UTP-induced AA release. 5. Short-term treatment with PMA (1 microM) inhibited the UTP-stimulated accumulation of IP and increase in [Ca2+]i, but had no effect on the release of AA. In contrast, the AA release caused by thapsigargin was increased by PMA. 6. The role of PKC in UTP- and thapsigargin-mediated AA release was shown by the blockade of these effects by staurosporine (1 microM), Ro 31-8220 (10 microM), Go 6976 (1 microM) and the down-regulation of PKC. 7. Following treatment of cells with SK&F 96365 (30 microM), thapsigargin-, but not UTP-, induced Ca2+ influx, and the accompanying AA release, were down-regulated. 8. Neither PD 98059 (100 microM), MEK a inhibitor, nor genistein (100 microM), a tyrosine kinase inhibitor, had any effect on the AA responses induced by UTP and thapsigargin. 9. We conclude that UTP-induced cPLA2 activity depends on the activation of PI-PLC and the sustained elevation of intracellular Ca2+, which is essential for the activation of cPLA2 by UTP and thapsigargin. The [Ca2+]i-dependent AA release that follows treatment with both stimuli was potentiated by the activity of protein kinase C (PKC). A pertussis toxin-sensitive pathway downstream of the increase in [Ca2+]i was also shown to be involved in AA release.     

Cyclosporin A inhibits apical secretory K+ channels in rabbit cortical collecting tubule principal cells

We used the cell-attached patch clamp configuration to examine the effect of basolateral cyclosporin A (CsA) exposure on low conductance K+ channels found in the principal cell apical membrane of rabbit cortical collecting tubule (CCT) primary cultures. Baseline K+ channel activity, measured as mean NPo (number of channels x open probability), was 2.7 +/- 1.1 (N = 29). NPo fell by 69% (0.84 +/- 0.32; N = 32) in cultures pretreated with 500 ng/ml CsA for 30 minutes prior to patching. Chelation of intracellular [Ca2+]i (10 mM BAPTA/AM; N = 8) or removal of extracellular Ca2+ (N = 9), but not prevention of [Ca2+]i store release (10 microM TMB-8; N = 7), abolished CsA-induced inhibition. This suggested that CsA effects were mediated by an initial rise in [Ca2+]i via Ca2+ influx. Either 25 nM AVP (N = 10) or 0.25 microM thapsigargin (N = 8) (causing IP3-dependent and -independent release of [Ca2+]i stores, respectively) augmented, while 25 pM (N = 6) or 250 pM AVP (N = 8) reversed CSA-induced channel inhibition. Apical membrane protein kinase C (PKC) activation with 0.1 microM phorbol ester, PMA (N = 8) or 10 microM synthetic diacylglycerol, OAG (N = 7), mimicked (mean NPo = 0.99 +/- 0.40) the inhibitory effect of CsA. Apical PKC inhibition by prolonged apical exposure to PMA (N = 10) or 100 microM D-sphingosine (N = 6) blocked CsA's effect. Cyclic AMP increasing maneuvers, 10 microM forskolin (N = 5) or 0.5 mM db-cAMP (N = 8), stimulated basal K+ channel activity in the absence of CsA. In Conclusion: (1) basolateral exposure to CsA inhibits the activity of apical membrane 13 pS channels responsible for physiologic K+ secretion in rabbit CCT principal cells. (2) The inhibition is mediated by changes in intracellular Ca2+ and activation of apical PKC. (3) Pharmacologic AVP (nM) augments CsA-induced inhibition by releasing intracellular Ca2+ stores; more physiologic AVP (pM) attenuates channel inhibition, probably through cAMP generation. (4) Inhibition of apical secretory K+ channels by CsA likely contributes to decreased kaliuresis and clinical hyperkalemia observed in patients on CsA therapy.     

Role of PKC in the effects of alpha1-adrenergic stimulation on Ca2+ transients, contraction and Ca2+ current in guinea-pig ventricular myocytes

The effects of alpha1-adrenoceptor stimulation on intracellular Ca2+ transients, contractility and L-type Ca2+ current (ICa,L) were studied in single cells isolated from ventricles of guinea-pig hearts. The aim of our study was to elucidate the mechanisms of the positive inotropic effect of alpha1-adrenergic stimulation by focussing on the role of protein kinase C (PKC). Phenylephrine, an alpha1-adrenergic agonist, at concentrations of 50-100 microM elicited a biphasic inotropic response: a transient negative inotropic response (22.9+/-6.0% of control) followed by a sustained positive inotropic response (61.0+/-8.4%, mean+/-SE, n=12). The Ca2+ transient decreased by 10.2+/-3.9% during the negative inotropic phase, while it increased by 67.7+/-10% (n=12) during the positive inotropic phase. These effects were inhibited by prazosin (1 microM), a alpha1-adrenergic antagonist. Phenylephrine increased the ICa,L by 60.8+/-21% (n=5) during the positive inotropic phase. To determine whether activation of PKC is responsible for the increases in Ca2+ transients, contractile amplitude and ICa,L during alpha1-adrenoceptor stimulation, we tested the effects of 4beta-phorbol 12-myristate 13-acetate (PMA), a PKC activator, and of bisindolylmaleimide I (GF109203X) and staurosporine, both of which are PKC inhibitors. PMA mimicked phenylephrine's effects on Ca2+ transients, contractile amplitude and ICa,L. PMA (100 nM) increased the Ca2+ transient, contractile amplitude and ICa,L by 131+/-17%, 137+/-25% (n=8), and 81.1+/-26% (n=5), respectively. Prior exposure to GF109203X (1 microM) or staurosporine (10 nM) prevented the phenylephrine-induced increases in Ca2+ transients, contractile amplitude and ICa,L. Our study suggests that during alpha1-adrenoceptor stimulation increase in ICa,L via PKC causes an increase in Ca2+ transients and thereby in the contractile force of the ventricular myocytes.     

Characterization of the calcium-activated chloride channel in isolated guinea-pig hepatocytes

Macroscopic and unitary currents through Ca(2+)-activated Cl- channels were examined in enzymatically isolated guinea-pig hepatocytes using whole-cell, excised outside-out and inside-out configurations of the patch-clamp technique. When K+ conductances were blocked and the intracellular Ca2+ concentration ([Ca2+]i) was set at 1 microM (pCa = 6), membrane currents were observed under whole-cell voltage-clamp conditions. The reversal potential of the current shifted by approximately 60 mV per 10-fold change in the external Cl- concentration. In addition, the current did not appear when Cl- was omitted from the internal and external solutions, indicating that the current was Cl- selective. The current was activated by increasing [Ca2+]i and was inactivated in Ca(2+)-free, 5 mM EGTA internal solution (pCa > 9). The current was inhibited by bath application of 9-anthracenecarboxylic acid (9-AC) and 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in a voltage-dependent manner. In single channel recordings from outside-out patches, unitary current activity was observed, whose averaged slope conductance was 7.4 +/- 0.5 pS (n = 18). The single channel activity responded to extracellular Cl- changes as expected for a Cl- channel current. The open time distribution was best described by a single exponential function with mean open lifetime of 97.6 +/- 10.4 ms (n = 11), while at least two exponentials were required to fit the closed time distributions with a time constant for the fast component of 21.5 +/- 2.8 ms (n = 11) and that for the slow component of 411.9 +/- 52.0 ms (n = 11). In excised inside-out patch recordings, channel open probability was sensitive to [Ca2+]i. The relationship between [Ca2+]i and channel activity was fitted by the Hill equation with a Hill coefficient of 3.4 and the half-maximal activation was 0.48 microM. These results suggest that guinea-pig hepatocytes possess Ca(2+)-activated Cl- channels.     

Angioscopy in vascular surgery: the state of the art

Incubation of a human fibrosarcoma cell line HT-1080 in increasing concentration of Ca2+ was found to enhance endocytic internalization of a fluid phase marker, horseradish peroxidase. At 16.8 mM Ca2+, generation of the effect required incubation for more than 45 min. The effect was reversed by removal of the excess ion for 30 min. Monitoring the intracellular concentration showed that the incubation induced a transient large Ca2+ influx followed by a recovery to 230 +/- 50 nM instead of the normal level of 83 +/- 5 nM. The activation was not inhibited by inhibitors of protein kinases nor a cAMP antagonist. In contrast, the effect was prevented by okadaic acid (OKA) at 100 nM without detectable effect on the basal activity. Fluid phase uptake by HT-1080 cells was also enhanced by phorbol 12-myristate 13-acetate (PMA). In contrast to the case with Ca2+, OKA at 100 nM did not prevent the PMA effect but further enhanced the endocytosis. The effect of OKA was concentration-dependent, as the reagent at 1 microM inhibited not only both the activation but also the basal activity. In Ca(2+)- or PMA-stimulated cells, FITC-dextran was delivered to endosomes that had been labeled with TRITC-transferrin. In contrast, following treatment with a combination of PMA and 100 mM OKA, fluid phase was internalized in vesicular compartments devoid of transferrin labeling. These results suggest that, through differential modifications of protein phosphorylation, endocytosis can be enhanced distinctively either by employing conventional receptor-bearing compartments or generating a new endosomal population.     

Nitric oxide (NO)-induced activation of large conductance Ca2+-dependent K+ channels (BK(Ca)) in smooth muscle cells isolated from the rat mesenteric artery

1. To assess the action of nitric oxide (NO) and NO-donors on K+ current evoked either by voltage ramps or steps, patch clamp recordings were made from smooth muscle cells freshly isolated from secondary and tertiary branches of the rat mesenteric artery. 2. Inside-out patches contained channels, the open probability of which increased with [Ca2+]i. The channels had a linear slope conductance of 212+/-5 pS (n = 12) in symmetrical (140 mM) K+ solutions which reversed in direction at 4.4 mV. In addition, the channels showed K+ selectivity, in that the reversal potential shifted in a manner similar to that predicted by the Nernst potential for K+. Barium (1 mM) applied to the intracellular face of the channel produced a voltage-dependent block and external tetraethylammonium (TEA; at 1 mM) caused a large reduction in the unitary current amplitude. Taken together, these observations indicate that the channel most closely resembled BK(Ca). 3. In five out of six inside-out patches, NO (45 or 67 microM) produced an increase in BK(Ca) activity. In inside-out patches, BK(Ca) activity was also enhanced in some patches with 100 or 200 microM 3-morpholino-sydnonimine (SIN-1) (4/11) and 100 microM sodium nitroprusside (SNP) (3/8). The variability in channel opening with the NO donors may reflect variability in the release of NO from these compounds. 4. In inside-out patches, 100 microM SIN-1 failed to increase BK(Ca) activity (in all 4 patches tested), while at a higher (500 microM) concentration SIN-1 had a direct blocking effect on the channels (n = 3). NO applied directly to inside-out patches increased (P < 0.05) BK(Ca) activity in two patches. 5. In the majority of cells (6 out of 7), application of NO (45 or 67 microM) evoked an increase in the amplitude of whole-cell currents in perforated patches. This action was not affected by the soluble guanylyl cyclase inhibitor, 1H-[1,2,4] oxadiazolo [4,3-a]quinoxalin-1-one (ODQ). An increase in whole-cell current was also evoked with either of the NO donors, SIN-1 or SNP (each at 100 microM). With SIN-1, the increase in current was blocked with the BK(Ca) channel blocker, iberiotoxin (50 nM). 6. With conventional whole-cell voltage clamp, the increase in the outward K+ current evoked with SIN-1 (50-300 microM) showed considerable variability. Either no effect was obtained (11 out of 18 cells), or in the remaining cells, an average increase in current amplitude of 38.7+/-10.2% was recorded at 40 mV. 7. In cell-attached patches, large conductance voltage-dependent K+ channels were stimulated by SIN-1 (100 microM) applied to the cell (n = 5 patches). 8. These data indicate that NO and its donors can directly stimulate BK(Ca) activity in cells isolated from the rat mesenteric artery. The ability of NO directly to open BK(Ca) channels could play an important functional role in NO-induced relaxation of the vascular smooth muscle cells in this small resistance artery.     

Erectile dysfunction of vasculogenic origin: ultrastructural evaluation of cavernous myo-endothelial unit as prognostic index of penile revascularization]

1. The present study examined the effects of various agents on high calcium-induced relaxation of the rabbit thoracic aorta precontracted by phenylephrine (0.1 microM) or KCl (30 mM). 2. The vascular smooth muscle relaxation caused by high calcium was not changed in the presence of endothelium, glibenclamide (3 microM), TEA (5 mM), verapamil (1 microM), lidocaine (0.1 mM) and vanadate (0.1 mM). 3. In the presence of ouabain (0.1 mM) or potassium-free medium, high calcium-induced relaxation was completely abolished. 4. When rings were precontracted by high concentrations of phenylephrine (1 microM, 10 microM) and KCl (30 mM, 45 mM, 60 mM), calcium-induced relaxation was gradually decreased. 5. A low concentration of calcium ionophore A-23187 (0.1 microM) did not change calcium-induced relaxation, but A-23187 at a high concentration (1 microM) depressed this relaxation. 6. These results suggest that Na-K-ATPase activation could be responsible for high calcium-induced relaxation.     

Multiple effects of nordihydroguaiaretic acid on ionic currents in rat isolated type I carotid body cells

1. The effects of the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA) on the ionic currents of rat carotid body type I cells were investigated by use of whole-cell and outside-out patch clamp techniques. 2. NDGA (5-50 microM) produced a concentration-dependent inhibition of whole-cell K+ currents at all activating test potentials (holding potential -70 mV). The time-course of the inhibition was also concentration-dependent and the effects of NDGA were only reversible following brief periods of exposure (<2 min). Another lipoxygenase inhibitor, phenidone (5 microM), was without effect on whole-cell K+ currents in carotid body type I cells. 3. NDGA (5-50 microM) also inhibited whole-cell Ca2+ channel currents (recorded with Ba2+ as charge carrier) in a concentration-dependent manner. 4. Isolation of voltage-gated K+ channels by use of high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions revealed a direct inhibition of the voltage-sensitive component of the whole-cell K+ current by NDGA (50 microM). 5. In excised, outside-out patches NDGA (20-50 microM) increased large conductance, Ca2+ activated K+ channel activity approximately 10 fold, an effect which could be reversed by either tetraethylammonium (10 mM) or charybdotoxin (30 nM). 6. It is concluded that NDGA activates maxi-K+ channels in carotid body type I cells and over the same concentration range inhibits voltage-sensitive K+ and Ca2+ channels. The inhibition of whole cell K+ currents seen is most likely due to a combination of direct inhibition of the voltage-sensitive K+ current and indirect inhibition of maxi-K+ channel activity through blockade of Ca2+ channels.     

Alterations of potassium channel activity in retinal Müller glial cells induced by arachidonic acid

Arachidonic acid, which is thought to be involved in pathogenetic mechanisms of the central nervous system, has been shown previously to modulate neuronal ion channels and the glutamate uptake carrier of retinal glial (Müller) cells. We have used various configurations of the patch-clamp technique to determine the effects of arachidonic acid on the K+ currents of freshly isolated Müller glial cells from rabbit and human. Arachidonic acid reduced the peak amplitude of the transient (A-type) outward K+ currents in a dose-dependent and reversible manner, with a 50% reduction achieved by 4.1 microM arachidonic acid. The inward rectifier-mediated currents remained unchanged after arachidonic acid application. The amplitude of the Ca(2+)-activated K+ outward currents (KCa), which were blocked by 1 mM tetraethylammonium chloride and 40 nM iberiotoxin, respectively, was dose-dependently elevated by bath application of arachidonic acid. The activation curve of the KCa currents shifted towards more negative membrane potentials. Furthermore, arachidonic acid was found to suppress inwardly directed Na+ currents. In cell-attached recordings with 3 mM K+ in the bath and 130 mM K+ in the pipette, the KCa channels of rabbit Müller cells displayed a linear current-voltage relation, with a mean slope conductance of 102 pS. In excised patches, the slope conductance was 220 pS (150 mM K+i/130 mM K+o). The opening probability of the KCa channels increased during membrane depolarization and during elevation of the free Ca2+ concentration at the intracellular face of the membrane patches. Bath application of arachidonic acid caused a reversible increase of the single-channel opening probability, as well as an increase of the number of open channels. Arachidonic acid did not affect the single-channel conductance. Since arachidonic acid also stimulates the KCa channel activity in excised patches, the action of arachidonic acid is assumed to be independent of changes of the intracellular calcium concentration. Our results demonstrate that arachidonic acid exerts specific effects on distinct types of K+ channels in retinal glial, cells. In pathological cases, elevated arachidonic acid levels may contribute to prolonged Müller cell depolarizations, and to the initiation of reactive glial cell proliferation.     

Fusion pore expansion in horse eosinophils is modulated by Ca2+ and protein kinase C via distinct mechanisms

Using the patch-clamp technique, we studied the role of protein phosphorylation and dephosphorylation on the exocytotic fusion of secretory granules with the plasma membrane in horse eosinophils. Phorbol 12-myristate 13-acetate (PMA) had no effect on the amplitude and dynamics of degranulation, indicating that the formation of fusion pores is insensitive to activation of protein kinase C (PKC). Fusion pore expansion, however, was accelerated approximately 2-fold by PMA, and this effect was abolished by staurosporine. Elevating intracellular Ca2+ to 1.5 microM also resulted in a 2-fold acceleration of pore expansion; this effect was not prevented by staurosporine, indicating that intracellular Ca2+ and activation of PKC accelerate fusion pore expansion via distinct mechanisms. However, fusion pores can expand fully even when PKC is inhibited. In contrast, the phosphatase inhibitor alpha-naphthylphosphate inhibits exocytotic fusion and slows fusion pore expansion. These results demonstrate that, subsequent to its formation, fusion pore expansion is under control of proteins subject to functional changes based on their phosphorylation states.     

Dual regulation of the Na+/H(+)-exchange in rat peritoneal mast cells: role of protein kinase C and calcium on pHi regulation and histamine release

1. The purpose of this study was to compare the actions of phorbol 12-myristate 13-acetate (PMA) and ionomycin on Na+/H+ exchange activation and histamine release to that of compound 48/80 in order to study the possible relationship between pHi and secretion of histamine in rat peritoneal mast cells. 2. Resting pHi in mast cells suspended in a bicarbonate-free physiological salt solution amounted to 6.73 +/- 0.05 (mean +/- s.d., n = 52). 3. PMA (20 nM) induced a substantial but rather slow increase in pHi. This response was very sensitive to inhibition by staurosporine, very sensitive to inhibition by 5-(N,N-hexamethylene)amiloride (HMA), insensitive to the absence of extracellular calcium (without EGTA), and sensitive to partial depletion of intracellular calcium with EGTA. 4. Ionomycin (1 microM) induced a biphasic change in pHi that was sensitive to inhibition by HMA, insensitive to staurosporine. In the absence of extracellular calcium using EGTA, the biphasic response disappeared, leaving only a slow, and diminished change in pHi. 5. The effects of ionomycin and PMA on pHi were additive. 6. Addition of the secretagogue compound 48/80 (1 microgram ml-1) increased pHi, substantially, delta pHi amounting to 0.29 +/- 0.05 pH-units (n = 4). The biphasic pHi-response was insensitive to the absence of extracellular calcium (without EGTA). The initial fast response in pHi was, however, inhibited by HMA, not staurosporine. 7. The finding that staurosporine and HMA each inhibited approximately half of the compound 48/80-induced pHi-response, whereas both inhibitors completely abolished the compound 48/80-induced pHi-response seems to indicate that two independent pathways for the activation of the Na+/H+ exchange were stimulated by compound 48/80. 8. The histamine release induced via both PKC activation (using PMA) and calcium (using ionomycin) were much larger than the sum of each activation pathway, whereas in the absence of extracellular calcium using EGTA, the histamine release in response to PMA and ionomycin was completely abolished. 9. The compound 48/80-induced histamine release was partially sensitive to inhibition by HMA (approximately 30% inhibition) and partially sensitive to inhibition by staurosporine (approximately 50% inhibition). Preincubation with staurosporine and HMA before stimulation with compound 48/80 showed the same degree of inhibition as observed after staurosporine alone, even though this combination of drugs completely inhibited the pHi-response. Furthermore, compound 48/80-induced histamine release was not dependent on the presence of extracellular calcium (with and without EGTA). 10. In spite of the similarities in second messenger pathways for pHi regulation and histamine release, it is, however, not very likely that these two processes are directly related. It is, however, possible, that an increase in pHi plays a permissive, rather than an essential role for histamine release in rat peritoneal mast cells. This hypothesis was supported by the finding that preincubation with the Na+/H+ exchange-inhibitor HMA inhibited 30% of the compound 48/80-induced histamine secretion.     

Potentiation and inhibition of nicotinic acetylcholine receptors by spermine in the TE671 human muscle cell line

Nicotinic acetylcholine receptors (nAChR) of the TE671 cell line were investigated using whole-cell and membrane patch recording techniques. At negative holding potentials (VH), pulses of acetylcholine (ACh) elicited whole-cell inward currents that rapidly desensitized. The EC50 value for ACh at VH = -60 mV was 7.8 microM. The ACh-induced current reversed at approximately 0 mV. Desensitization of nAChR by ACh was biphasic and reversible within approximately 20 sec. Spermine (1-100 microM) potentiated responses to ACh (10 microM - 1 mM) by reducing the rate of onset of desensitization; potentiation was inhibited by arcaine (10-100 microM). Spermine (1 mM) noncompetitively antagonized the AChinduced current. Antagonism by 1 to 5 mM spermine was voltage-dependent, increasing with negative VH. In 100 microM arcaine, this antagonism was shown to contain a voltage-independent component. Spermine (10 mM) increased the EC50 values for ACh, suggesting that at this concentration the polyamine is also a competitive antagonist. Single channel openings elicited during application of ACh to outside-out patches had a conductance of 47 pS at VH = -60 mV. At 10 and 100 microM, spermine increased channel open probability (po), but at 1 mM spermine, po was not significantly different from controls. The single channel conductance for ACh was unaffected by 10 and 100 microM spermine, but was decreased by 1 mM spermine. Spermine promoted the occurrence of approximately 27 pS openings. It is proposed that spermine acts at an excitatory modulatory site similar to that present on N-methyl-D-aspartate receptors and at least three inhibitory sites on nAChR of TE671 cells.     

A1 adenosine receptors modulate respiratory activity of the neonatal mouse via the cAMP-mediated signaling pathway

The effects of adenosine and its analogs on the function of the respiratory center were studied in the spontaneously active rhythmic slice of neonatal and juvenile mice (4-14 days old). Whole cell, spontaneous postsynaptic currents (sPSCs) and single channel KATP currents were recorded in inspiratory neurons of the pre-B?tzinger complex. Adenosine (50-600 microM) inhibited the respiratory rhythm. This was accompanied by increase in the activity of KATP channels in cell-attached patches. The A1 adenosine receptor agonist, 2-chloro-N6-cyclopentyladenosine (CCPA, 0.3-2 microM), inhibited the respiratory rhythm, sPSCs, and enhanced activity of KATP channels. The A1 adenosine receptor antagonist, 8-cyclopentyl-1, 3-dipropylxanthine (DPCPX, 1-3 microM), showed opposite effects and occluded the CCPA actions. Agents specific for A2 adenosine receptors (CGS 21860 and NECA, both applied at 1-10 microM) were without effect. Elevation of intracellular cAMP concentration ([cAMP]i) by 8-Br-cAMP (200-500 microM), forskolin (0.5-2 microM), or isobutylmethylxantine (IBMX, 30-90 microM) reinforced the rhythm, whereas NaF (100-800 microM) depressed it. The open probability of single KATP channels in cell-attached patches decreased after application of forskolin and increased in the presence of NaF. [cAMP]i elevation reversed the effects of A1 receptors both on the respiratory rhythm and KATP channels. A1 receptors and [cAMP]i modified the hypoxic respiratory response. In the presence of A1 agonists the duration of hypoxic augmentation shortened, and depression of the respiratory rhythm occurred earlier. Elevation of [cAMP]i prolonged augmentation and delayed the development of the depression. We conclude that A1 adenosine receptors modulate the respiratory rhythm via inhibition of intracellular cAMP production and concomitant activation of KATP channels.     

Dual regulation of cerebrovascular tone by UTP: P2U receptor-mediated contraction and endothelium-dependent relaxation

1. The mechanisms of vascular tone regulation by extracellular uridine 5'-triphosphate (UTP) were investigated in bovine middle cerebral arterial strips. Changes in cytosolic Ca2+ concentration ([Ca2+]i) and force were simultaneously monitored by use of front-surface fluorometry of fura-2. 2. In the arterial strips without endothelium, UTP (0.1 microM-1 mM) induced contraction in a concentration-dependent manner. However, when the endothelium was kept intact, cumulative application of UTP (0.1-100 microM) (and only at 1 mM) induced a modest phasic contraction in arterial strips. This endothelium-dependent reduction of the UTP-induced contraction was abolished by 100 microM N omega-nitro-L-arginine (L-NOARG) but not by 10 microM indomethacin. In the presence of intact endothelium, UTP (30 microM) induced a transient relaxation of the strips precontracted with 30 nM U-46619 (a stable analogue of thromboxane A2), which was completely inhibited by pretreatment with L-NOARG but not with indomethacin. 3. In the endothelium-denuded strips, the contractile response to UTP was abolished by desensitization to either ATP gamma S or ATP (P2U receptor agonists), but not by desensitization to alpha, beta-methylene-ATP (P2x receptor agonist) or to 2-methylthio-ATP (P2Y receptor agonist). Desensitization to UTP abolished the contractile response to ATP. 4. In the endothelium-denuded artery, a single dose application of UTP induced an initial transient, and subsequently lower but sustained increase in [Ca2+]i and force. In the absence of extracellular Ca2+, UTP induced only the initial transient increases in [Ca2+]i and force, while the sustained increases in [Ca2+]i and force were abolished. UTP (1 mM) had no effect on the basic [Ca2+]i-force relationship obtained on cumulative application of extracellular Ca2+ at steady state of 118 mM K(+)-depolarization-induced contraction. 5. We conclude that in the presence of an intact endothelium, UTP-induced relaxation of preconstricted middle cerebral artery is mainly mediated indirectly, by the production of an endothelium-derived relaxing factor, but at high doses of UTP, vascular smooth muscle contraction is mediated directly via activation of P2U purinoceptor and [Ca2+]i elevation without Ca(2+)-sensitization of the contractile apparatus. UTP may thus exert a dual regulatory effect upon cerebrovascular tone, but in cases where the endothelium is impaired, it may also act as a significant vasoconstrictor.     

Endothelin receptor-mediated Ca2+ mobilization and contraction in bovine oviductal arteries: comparison with noradrenaline and potassium

1. The effects of endothelin-1 (ET-1) were studied in bovine oviductal arteries and compared to those of noradrenaline (NA) and high K+ (K+). The influence of endothelium, the receptor subtypes involved, and the mechanisms of Ca2+ mobilization were assessed. 2. ET-1 (0.1-300 nM) induced concentration-dependent contractions with a potency of 10(3) and 10(2) times higher than NA (0.1 microM-0.1 mM) and K+ (9.5-119 mM), respectively. Removal of endothelium or NG-nitro-L-arginine (L-NOARG, 0.1 mM) pretreatment did not affect responses to either ET-1 or K+, whereas the NA response was significantly increased. Indomethacin (1 microM) had no effect on either of these agonists. 3. The rank order of potency for the ET isopeptides was: ET-1 = ET-2 > ET-3. The ETA receptor-selective agonist, sarafotoxin 6c (S6c), had no effect. The ETA receptor-selective antagonist, BQ-123, showed a competitive antagonism on the ET-1 response (pA2 value of 6.58 +/- 0.01), whereas contractions to ET-3 were completely abolished by BQ-123 at 0.1 microM. 4. Concentration-response curves to both ET-1 and NA were shifted to the right and their maximum response reduced to approximately 56% and 65% of controls, respectively, under 30 min of incubation in Ca(2+)-free solution, whereas responses to K+ were almost abolished by this treatment. Contractions to both NA (30 microM) and ET-1 (30 nM) were maximally inhibited after 10 min of extracellular Ca2+ deprivation. 5. Contractions to ET-1 were more potently inhibited by nickel (Ni2+, 0.3 mM), whereas nifedipine (1 microM) and cadmium (Cd2+, 0.1 mM) induced only a slight effect. In contrast, opposite effects were found for both NA and K+. 6. Treatment with ryanodine (100 microM) and caffeine (10 mM) in Ca(2+)-free solution reduced the tension measured 5 min after NA (30 microM) and ET-1 (30 nM) addition, but the sustained response (tension at 25 min) remained unaffected. 7. Calphostin C (1 microM), a specific protein kinase C (PKC) inhibitor, reduced the maximum contractile response to ET-1 by about 50% without significantly affecting its pD2 value. 8. These results suggest that ET-1 acts in bovine oviductal arteries by directly activating a homogenous population of ETA receptors in smooth muscle, without endothelial modulation. Several Ca2+ activation mechanisms seem to be involved in the contractile action of the peptide, including: (1) extracellular Ca2+ entrance through Ni(2+)-sensitive and L-type Ca2+ channels; (2) intracellular Ca2+ release from a ryanodine-sensitive Ca2+ store; and (3) sensitization of the contractile machinery to Ca2+ via PKC.     

Adenosine A1 receptor-mediated inhibition of evoked glutamate release is coupled to calcium influx decrease in goldfish brain synaptosomes

Binding of [3H]cyclohexyladenosine (CHA) to the cellular fractions and P2 subfractions of the goldfish brain was studied. The A1 receptor density was predominantly in synaptosomal membranes. In goldfish brain synaptosomes (P2), 30 mM K+ stimulated glutamate, taurine and GABA release in a Ca(2+)-dependent fashion, whereas the aspartate release was Ca(2+)-independent. Adenosine, R-phenylisopropyladenosine (R-PIA) and CHA (100 microM) inhibited K(+)-stimulated glutamate release (31%, 34% and 45%, respectively). All of these effects were reversed by the selective adenosine A1 receptor antagonist, 8-cyclopentyltheophylline (CPT). In the same synaptosomal preparation, K+ (30 mM) stimulated Ca2+ influx (46.8 +/- 6.8%) and this increase was completely abolished by pretreatment with 100 nM omega-conotoxin. Pretreatment with 100 microM R-PIA or 100 microM CHA, reduced the evoked increase of intra-synaptosomal Ca2+ concentration, respectively by 37.7 +/- 4.3% and 39.7 +/- 9.0%. A possible correlation between presynaptic A1 receptor inhibition of glutamate release and inhibition of calcium influx is discussed.     

Effects of the functional elimination of sarcoplasmic reticulum on the manganese-dependent norepinephrine-induced contractions of the guinea pig vas deferens

We investigated the effects of inhibitors of the sarcoplasmic reticulum (SR) functions on the tonic contractions induced by norepinephrine (NE) in the Ca(2+)-depleted Mn(2+)-loaded vas deferens of the guinea pig in the absence of both Ca2+ and Mn2+ (Mn(2+)-dependent NE-contraction). In control preparations without Ca(2+)-depletion and Mn(2+)-loading, either cyclopiazonic acid (CPA, 10 microM) or ryanodine (RYA, 3 microM) inhibited the initial phasic and tonic components but not the large phasic component of NE-induced contraction in normal medium containing 2.2 mM Ca2+. In contrast, CPA did not affect the Mn(2+)-dependent NE-contractions. The inhibitory effect of RYA slowly developed with each repetition of the Mn(2+)-dependent NE-contraction and the magnitude of the inhibition was slight. A23187 (10 microM) inhibited the NE-induced contractions of the control preparations in the same manner as CPA and RYA. Although A23187 did not induce contractions in the Mn(2+)-loaded preparations, A23187 augmented the Mn(2+)-dependent NE-contractions. The augmented tonic contractions returned to the resting level by washing NE and A23187. The augmentation remained for 3 successive contractions in the absence of A23187. However, the 2nd application of A23187 did not augment the contraction. These results suggest that neither Mn(2+)-release from SR nor Mn(2+)-influx from the extracellular space contributes to the Mn(2+)-dependent NE-contractions. We concluded that NE induces Mn(2+)-dependent contractions by increasing Mn2+ sensitivity of contractile processes but not by increasing intracellular Mn2+ concentration.