Tyrosine protein kinase inhibitors prevent activation of cardiac swelling-induced chloride current

The effect of tyrosine protein kinase inhibitors on the swelling-induced chloride current (ICl-swelling) of dog atrial myocytes was studied using the whole-cell patch-clamp recording technique. Currents were measured during hyperpolarizing voltage ramps with potassium currents blocked by cesium. Osmolarity was varied using mannitol. Exposure to hypotonic solution (approximately 249 mosmol/kg) activated ICl-swelling. Hypertonic solution (approximately 363 mosmol/kg) was used to shrink swollen cells and turn off ICl-swelling. In studies on the acute effect of tyrosine protein kinase inhibitors each cell was swollen three separate times. Control, treatment, and washout ICl-swelling were compared. Genistein (50-80 microM) prevented reactivation of ICl-swelling without affecting cell size. The effect of genistein partially subsided upon washout. The effect of genistein on ICl-swelling was not mimicked by 80 microM daidzein, a related compound that does not inhibit tyrosine protein kinases. When intracellular adenosine 5'-O-(3-thiotriphosphate (ATP[gamma S]) was used, genistein did not prevent the reactivation of ICl-swelling. Intracellular ATP[gamma S] did not result in a persistent activation of ICl-swelling when cell size was returned to control. Acute exposure to 1 microM herbimycin A or 100 microM tyrphostin 51 did not prohibit the activation of ICl-swelling. A 24-h exposure to 1 microM herbimycin A did inhibit ICl-swelling. The results provide important clues regarding the activation mechanism for ICl-swelling and suggest that a tyrosine protein phosphorylation may be necessary, but not sufficient, for activation of ICl-swelling.     

Protein kinase C inhibitors block amphetamine-mediated dopamine release in rat striatal slices

The stimulant drug amphetamine is postulated to enhance dopamine release through the plasmalemmal dopamine transporter by an exchange diffusion with synaptosomal dopamine. Because protein kinase C has been shown to have an effect on dopamine transporter activity, we examined the effect of protein kinase C inhibitors on endogenous dopamine release stimulated by amphetamine in perfused rat striatal slices. At concentrations of 1 microM, the selective protein kinase C inhibitors chelerythrine, Ro31-8220 and calphostin C nearly completely inhibited endogenous dopamine release elicited by 1 microM amphetamine. The inactive analog bisindoylmaleimide V had no effect. Extracellular Ca++ was not required for the effect of the inhibitors. The importance of vesicular dopamine release was examined by determining inhibitor activity in reserpine-treated rats. Dopamine release elicited by 1 microM amphetamine was not significantly altered in reserpine-treated rats compared with control animals. Ro31-8220 at 1 microM completely blocked amphetamine-induced dopamine release in reserpine-treated rats. Activation of protein kinase C with 250 nM of the phorbol ester 12-O-tetradecanoylphorbol 13-acetate increased dopamine release, and the release was not additive with 1 microM amphetamine. Both chelerythrine and Ro31-8220 at 1 microM increased [3H]dopamine uptake by 17% and 30%, respectively, whereas a brief exposure to 12-O-tetradecanoylphorbol 13-acetate slightly inhibited [3H]dopamine uptake. Our results suggest that amphetamine-mediated dopamine release through the plasmalemmal transporter is highly dependent on protein kinase C activity.     

Involvement of L-type-like amino acid transporters in S-nitrosocysteine-stimulated noradrenaline release in the rat hippocampus

Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S-nitroso-L-cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [3H]NA release from prelabeled hippocampal slices in a dose-dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na+-containing Tyrode's buffer, SNC-stimulated [3H]NA release was inhibited 30% by the coaddition of L-leucine. In the Na+-free, choline-containing buffer, SNC-stimulated [3H]NA release, which was similar to that in the Na+-containing buffer, was inhibited markedly by L-leucine, L-alanine, L-methionine, L-phenylalanine, and L-tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of L-leucine was stronger than that of D-leucine. A specific inhibitor of the L-type amino acid transporter, 2-aminobicyclo[2.2.1]-heptane-2-carboxylate (BCH), inhibited the effects of SNC on [3H]NA release in the Na+-free buffer. Uptake of L-[3H]leucine into the slices in the Na+-free buffer was inhibited by SNC, BCH, and L-phenylalanine, but not by L-lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by L-leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 microM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L-type-like amino acid transporter, at least in Na+-free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP-independent manner.     

Protein kinase A increases the tension cost and unloaded shortening velocity in skinned rat cardiac muscle

To address controversies concerning the effect of beta-adrenergic stimulation on the rate of cross-bridge cycling in cardiac muscle, we measured ca(2+)-induced isometric tension development, unloaded shortening velocity (Vmax) and ATPase activity of demembranated (Triton X-100 skinned) rat right ventricular trabeculae before and after treatment with the catalytic subunit of protein kinase A (PKA), which is known to mimic the action of beta-adrenergic agonists in demembranated preparations. PKA treatment (1 U/microliter, 40 min) shifted the pCa-tension relation to the right from 5.41 to 5.26 at pCa50 (the [Ca2+] required for half maximal steady state tension) without changing the steepness of the pCa-tension relation and the maximum Ca(2+)-activated tension; Vmax, as determined by the slack test, was increased for a given pCa value, despite the reduced level of isometric tension. PKA treatment also shifted the pCa-ATPase activity to the right slightly from 5.47 to 5.40 at pCa50 (the [Ca2+] required for half maximal ATPase activity), but increased the ATPase activity during a given level of steady isometric tension generation, resulting in a 33% increase of the tension cost (ATPase activity/tension). All the results obtained strongly suggest that, in rat right ventricular trabeculae, beta-adrenergic stimulation may increase the rate of cross-bridge cycling by increasing the rate of cross-bridge detachment from actin through a PKA-mediated mechanism, although PKA reduces the Ca(2+)-sensitivity of the contractile system.     

Phospholipase C inhibitors attenuate arrhythmias induced by kappa-receptor stimulation in the isolated rat heart

To determine whether the phospholipase C (PLC)/inositol 1,4,5 trisphosphate (IP3)/Ca2+ pathway mediates cardiac arrhythmias induced by kappa-opioid receptor stimulation, the effects of U50,488H, a selective kappa-opioid receptor agonist, on cardiac rhythm in a isolated perfused rat heart, intracellular calcium ([Ca2+]i) in a single ventricular myocyte and IP3 production in myocytes were studied in the presence and absence of PLC inhibitors. U50,488H, the effects of which had been shown to be abolished by a selective kappa-receptor antagonist, nor-binaltorphimine, induced arrhythmias dose-dependently and increased both [Ca2+]i and IP3-production in the heart. More importantly, the effects of U50,488H were blocked by PLC inhibitors, neomycin and streptomycin. To further confirm the selectivity of action of the PLC inhibitor, the effects of another PLC inhibitor U73122 and its inactive structural analog, U73343, on cardiac rhythm in the isolated perfused rat heart were compared. The former did, while the latter did not, block the arrhythmogenic effect of U50,488H. We also determined whether the effects of kappa-receptor stimulation involves a pertussis toxin (PTX)-sensitive G-protein. We found that pretreatment with PTX at 4 microg/l for 10 min, a treatment shown to affect PTX sensitive G-protein-mediated functions, attenuated significantly the U50,488H-induced arrhythmias. The present study provides evidence that kappa-receptor stimulation-induced cardiac arrhythmias involves, at least partly, the PLC/IP3/Ca2+ pathway as well as a PTX sensitive G-protein.     

Transmitter amino acid release from rat neocortex: complete versus incomplete ischemia models

Release of the excitotoxic amino acids, glutamate and aspartate, from the ischemic rat cerebral cortex was compared in two models; the seven vessel occlusion model (7VO) of complete cerebral ischemia and the four vessel occlusion model (4VO) of incomplete cerebral ischemia. Amino acid efflux into cortical superfusates was measured using cortical cups placed on both hemispheres. Whereas a 20 min period of ischemia causes a pronounced release of glutamate and aspartate from the 4VO model, efflux was significantly reduced in the 7VO model. Release of the inhibitory transmitter GABA, was similar in the two models. This result suggests that excitotoxic amino acid efflux into the extracellular spaces of the cerebral cortex may be enhanced by the residual blood flow in an incomplete ischemia.     

Protein kinase C in rat cerebral microvessels

Activation of protein kinase C is a key event in the transduction of receptor-mediated extracellular signals. Little is known about the role of protein kinase C in the microcirculation of the brain. In this study, we examined protein kinase C in isolated cerebral microvessels. A technique for partial purification of protein kinase C from microvessels was employed, using Q-Sepharose batch adsorption and single-step salt elution in microfuge tubes. This procedure greatly reduced variability and increased protein kinase C specific activity in both the cytosolic and particulate fractions by nearly 50-fold. The identity of the enzyme was confirmed by its inhibition by staurosporine and bisindolylmaleimide and by its translocation in response to phorbol ester. The level of protein kinase C was assessed by [3H]phorbol ester binding and the endogenous substrates evaluated by in vitro phosphorylation studies. Finally, western blot analysis of protein kinase C isoforms indicated that the beta-isoform was present in both cytosolic and particulate fractions. The alpha-isoform was present at low levels in the cytosolic fraction, whereas the gamma-isoform was not detected.     

Effect of spinal kainic acid receptor activation on spinal amino acid and prostaglandin E2 release in rat

Current work has shown that spinal excitatory amino acid receptor activation can evoke physiological phenomena that may be mediated by the subsequent depolarization of glutamate-containing neurons and the activation of cyclo-oxygenase systems. To investigate this phenomenon, rats were implanted with lumbar intrathecal loop dialysis catheters for perfusion and an additional lumbar intrathecal PE-10 catheter for drug delivery. Two days after implantation, kainic acid (1 microgram) was injected intrathecally under light (0.5%) halothane anaesthesia and the spinal release of several amino acids and prostaglandin E2 was examined. Resting concentrations (mean expressed as pmol/25 microliters) of glutamate (89), aspartate (9), serine (387), glycine (597), taurine (185), asparagine (113) and prostaglandin E2 (0.43) were observed. Intrathecal kainic acid produced significant signs of arousal in the rat and evoked a significant increase (mean +/- S.E.M. of % baseline concentration) in aspartate (445 +/- 127%) and glutamate (221 +/- 35%). Prostaglandin E2 concentration was increased in the second post-injection sample (180 +/- 36%). Intrathecal pretreatment with 6-cyano-7-nitroquinoxaline-2, 3-dione (3 micrograms or 10 micrograms), a non-N-methyl-D-aspartate receptor antagonist, blocked amino acid but not prostaglandin E2 release after kainic acid injection. Pretreatment with MK-801 (10 micrograms; non-competitive NMDA receptor antagonist) had no significant effect on evoked release of amino acids or prostaglandin E2. Indomethacin (10 micrograms, a cyclo-oxygenase inhibitor) pretreatment significantly decreased baseline prostaglandin E2 release in control animals (61 +/- 6%) and suppressed kainic acid-evoked aspartate, taurine and prostaglandin E2 release, but had no effect on the concentration of glutamate after kainic acid injection. These data suggest that activation of spinal kainic acid receptors provides a powerful stimulus for secondary excitatory amino acid release and, consistent with the concurrent appearance of prostaglandin E2, that this release is potentiated by the release of a cyclo-oxygenase product.     

Prenatal ethanol exposure diminishes activity-dependent potentiation of amino acid neurotransmitter release in adult rat offspring

Prenatal ethanol exposure has been associated with long-lasting intellectual impairments in children. Previous studies suggest that these deficits are, in part, linked to neurochemical abnormalities that reduce the ability to sustain long-term potentiation (LTP) in hippocampal formation of adult offspring. One presynaptic component of LTP that manifests during the first half-hour after tetanic stimulation is an enhancement of amino acid neurotransmitter release. Given that the onset of enhanced neurotransmitter release correlates temporally with the decay of hippocampal LTP in prenatal ethanol-exposed offspring, we tested the hypothesis that prenatal ethanol exposure reduces tetanus stimulus-induced potentiation of electrically evoked amino acid release in hippocampal slices. Rat dams consumed 1 of 3 diets throughout gestation: (1) a BioServ liquid diet containing 5% (v/v) ethanol (26% ethanol-derived calories) that produces a maternal peak blood ethanol concentration of 83 mg/dl; (2) pair-fed an isocalorically equivalent amount of 0% ethanol liquid diet; or (3) Purina rat chow ad libitum. Hippocampal slices were prepared from adult offspring from each experimental diet group. Neither the amount of hippocampal slice tissue protein nor the incorporation of [3H]-D-aspartate (D-ASP) was affected by prenatal ethanol exposure. Furthermore, spontaneous efflux and electrically evoked D-ASP release were similar among the three diet groups. However, tetanus stimulus-induced potentiation of evoked D-ASP release in prenatal ethanol-exposed offspring was reduced to about one-third of the potentiation of D-ASP release observed in the control diet groups. These results suggest that prenatal ethanol exposure produces long-lasting deficits in the neurochemical mechanisms responsible for activity-dependent potentiation of amino acid transmitter release without affecting the synaptic machinery responsible for amino acid uptake, storage, and release.     

Protein and amino acid metabolism in chronic renal failure

We investigated the inhibitory effect of clarithromycin, a 14-membered ring macrolide antibiotic, on tumor-induced angiogenesis in vivo using a mouse dorsal air sac model. The inhibitory effect of clarithromycin was dose-dependent, and 100 mg/kg of clarithromycin administered intraperitoneally twice a day reduced the area of dense capillary network to about 30% that of the control. However, in concentrations up to 50 microM clarithromycin had no effect on lung cancer cells and human vascular endothelial cell growth, endothelial cell migration, or lung cancer cell production of the angiogenesis-inducing factors interleukin-8 and vascular endothelial growth factor. Clarithromycin in concentrations greater than 10 microM inhibited endothelial cell tube formation on Matrigel in a dose-dependent manner. These data suggest clarithromycin is a potent inhibitor of tumor-induced angiogenesis that exerts its effect by inhibiting endothelial cell tube formation, and may be a possible candidate for therapeutic application.     

Protein tyrosine kinase inhibitors reduce high-voltage activating calcium currents in CA1 pyramidal neurones from rat hippocampal slices

We have investigated the effects of protein tyrosine kinases (PTKs) inhibitors on high-threshold voltage activating (HVA) calcium currents in CA1 pyramidal neurones, whole-cell patch-clamp recorded from rat hippocampal slices. Genistein (100 microM) and tyrphostin B42 (100 microM), two PTKs inhibitors, reduced the steady-state barium current (IBa). On the other hand, daidzein and genistin (100 microM), two inactive analogues of genistein, had no effect on IBa amplitude. The inhibition induced by genistein was more pronounced at negative potentials. In order to characterize the calcium channels subtypes inhibited by PTKs inhibitors, we examined the effect of genistein in the presence of different calcium channel blockers. When L-type calcium channels were blocked by nifedipine, genistein induced a strong inhibition of the nifedipine-resistant IBa, suggesting an effect on non-L-type channels. Genistein did not antagonize the depressant effect of omega-Conotoxin-GVIA, a selective N-type calcium channel blocker, suggesting that N-type channels were not blocked by genistein. omega-Conotoxin-MVIIC (3-10 microM), a selective P/Q-type calcium channel blocker, greatly antagonized the depressant effect of genistein. Our results suggest that PTKs inhibitors reduce P-/Q-type, but not L- or N-types calcium currents in neurones of the CNS. The possible modulation of calcium channels by endogenous PTKs is discussed.     

Protein phosphatase inhibitors induce the release of serotonin from rat basophilic leukemia cells (RBL-2H3)

Oxidation capacities of laccase, manganese peroxidase (MnP) and lignin peroxidase (LiP) from Phlebia radiata were compared using non-phenolic (veratryl alcohol and ABTS) and phenolic (syringaldazine, vanillalacetone and Phenol red) compounds as reducing substrates. The effect of Mn(II) on enzyme reactions was also studied. Highest specific activities were recorded with laccase in the oxidation of phenolic compounds or ABTS and irrespective of Mn(II) concentration. LiP and MnP oxidized all these substrates but only the catalysis of MnP was dependent upon Mn(II). Only LiP clearly oxidized veratryl alcohol. However, Mn(II) interfered with this reaction by repressing veratraldehyde formation. These results point to multiple participation of manganese ions, either as a reducing (Mn(II)) or oxidizing (Mn(III)) agent in the enzymatic reactions.     

Protein kinase C inhibitors block generation of anoxia-induced long-term potentiation

The aim of this study was to study the possible intracellular mechanisms underlying the anoxia-induced long-term potentiation (anoxic LTP) in the CA1 neurons of rat hippocampal slices using extra- and intracellular recording techniques. Superfusion of the hippocampal slices with the protein kinase C (PKC) inhibitors NPC-15437 (20 microM) or H-7 (20 microM) specifically prevented the induction of anoxic LTP. Moreover, the anoxic LTP was completely abolished in neurons intracellularly recorded with the selective PKC inhibitor PKCI 19-36 (50 microM). The specific cAMP-dependent protein kinase (PKA) inhibitor Rp-cyclic adenosine 3',5'-monophosphate (Rp-cAMPS, 25 microM) had no effect on the anoxic LTP. It is concluded that induction of anoxic LTP requires the activation of postsynaptic PKC.     

Regulation of branched-chain amino acid metabolism in the lactating rat

There is evidence that during lactation, uptake of the essential branched-chain amino acids (BCAA) by mammary glands exceeds their output in milk protein. In this study, we have measured the potential of lactating rats to catabolize BCAA. The activity, relative protein and specific mRNA levels of the first two enzymes in the BCAA catabolic pathway, branched-chain aminotransferase (BCAT) and branched-chain alpha-keto acid dehydrogenase (BCKD), were measured in mammary gland, liver and skeletal muscle obtained from rat dams at peak lactation (12 d), from rat dams 24 h after weaning at peak lactation and from age-matched virgin controls. Western analysis showed that the mitochondrial BCATm isoenzyme was found in mammary gland. Comparison of lactating and control rats revealed that tissue BCATm activity, protein and mRNA were at least 10-fold higher in mammary tissue during lactation. Values were 1.3- to 1. 9-fold higher after 24 h of weaning. In mammary gland of lactating rats, the BCKD complex was fully active. In virgin controls and weaning dams, only about 20% of the complex was in the active state. Hypertrophy of the liver and mammary gland during lactation resulted in a 73% increase in total oxidative capacity in lactating rats. The results are consistent with increased expression of the BCATm gene in the mammary gland during lactation, whereas oxidation appears to be regulated primarily by changes in activity state (phosphorylation state) of BCKD.     

Delayed activation of cardiac swelling-induced chloride current after step changes in cell size

INTRODUCTION: A lag phase has been reported for the activation of cardiac swelling-induced chloride currents. Prior demonstrations of this lag used methods that produce gradual changes in cell size, making interpretation and quantification of the time course problematic. METHODS AND RESULTS: Isolated dog atrial cells were studied using the whole cell, patch clamp technique. Step changes in cell size were produced by application of transient pulses of positive pressure, and the time course for activation of the swelling-induced chloride current was observed. There was a distinct temporal dissociation between size changes and current activation that was temperature sensitive. Activation half-times were 98 +/- 31 seconds and 586 +/- 112 seconds at 36 degrees C and room temperature, respectively. Swelling-induced chloride currents were evoked in a higher percentage of cells at 36 degrees C (83%) compared with room temperature (50%). CONCLUSION: Cardiac swelling-induced chloride current activates with a distinct lag after step changes in cell size. The activation time course is temperature sensitive. These observations are consistent with the notion that signal transduction events, and not simply membrane stretch, are required for the activation of cardiac swelling-induced chloride current.     

Interleukin-1 stimulates hypothalamic inhibitory amino acid neurotransmitter release

In order to get a better insight into the function of amino acid residues located in the second transmembrane domain of the cystic fibrosis transmembrane conductance regulator (CFTR) protein, all exon 18 mutations found in cystic fibrosis (CF) patients were characterized at the protein and at the electrophysiological level. Of the different mutations present in transmembrane helix 12 (M1137V, M1137R, I11139V and deltaM1140), and the intracytoplasmic loop connecting TM12 and NBD2 (D1152H and D1154G), only M1137R interfered with the proper maturation of the protein. Permeability studies performed after injection of the different wild-type and mutant cRNAs in Xenopus laevis oocytes indicated that the mutations did not alter the permeability sequence of the CFTR channels. The whole cell cAMP activated chloride currents, however, were significantly reduced for M1137V, I1139V, D1152H and D1154G and close to zero for deltaM1140, indicating that these mutations interfere with the proper gating of the chloride channels.     

Preoptic rather than mediobasal hypothalamic amino acid neurotransmitter release regulates GnRH secretion during the estrogen-induced LH surge in the ovariectomized rat

Inhibitory and excitatory amino acid neurotransmitters have been suggested to participate in the feedback actions of estradiol (E2) on LH secretion. In the rat estrogen-receptive neurons have been demonstrated in the preoptic/anterior hypothalamic area (POA) and mediobasal hypothalamus/median eminence (MBH) and many of these neurons utilize gamma-aminobutyric acid (GABA) as neurotransmitter. The actions of excitatory amino acids (EAA) differ in ovariectomized (ovx) and ovx E2-substituted rats indicating that EAAs also participate in the positive feedback action of E2 on LH release. However, little information is available as to whether in vivo these transmitters exert their effects in the POA, where most of the GnRH perikarya are located, or in the MBH, i.e. at the nerve terminals. Therefore we conducted push pull cannula perfusions to compare the release rates of GABA, aspartate (ASP) and glutamate (GLU) in the MBH and POA. A subcutaneous implant of a silastic tube containing E2 resulted in LH surges in the afternoon of all treated animals. Prior to and during this LH surge the MBH release rates of neither GABA nor ASP nor GLU were significantly altered. In contrast, a conspicuous drop in preoptic GABA release occurred prior to and during the time of estrogen-induced LH surges and this was accompanied by enhanced preoptic secretion of ASP and GLU. In conclusion, we present the first data about amino acid release in the MBH during the E2-induced LH surge. Since only in the POA the LH surge is associated with changes in amino acid release, it appears that both inhibitory and excitatory amino acids act at the level of the GnRH cell bodies and/or dendrites and not on GnRH nerve terminals to mediate the feedback mechanism of E2 on LH release.     

The amino acid composition of rat bile

The pattern of amino acids in the bile of rats differs from the pattern in the serum of these animals, since bile contains significantly greater amounts of acidic and sulphur-containing amino acids and glycine than serum, while the serum contained more basic amino acids than bile, indicating that secretion of amino acids into bile may involve specific transport processes.     

Cardiac afferents attenuate the muscle metaboreflex in the rat

The influence of cardiac afferents on the muscle metaboreflex was examined in 16 rats instrumented with a Silastic-tipped catheter in the pericardial space and right atrium, Doppler ultrasonic flow probe and a pneumatic vascular occluder around the terminal aorta, and a Teflon catheter in the thoracic aorta. In protocol I (cardiac efferent and afferent blockade), the muscle metaboreflex was examined under three experimental conditions: 1) control, 2) cardiac autonomic efferent blockade [intrapericardial methylscopolamine (10 micrograms/kg) and propranolol (50 micrograms/kg)], and 3) combined cardiac autonomic efferent and afferent blockade (intrapericardial procainamide, 2%). In protocol II (blood volume expansion), the muscle metaboreflex was examined before and after 15% blood volume expansion. Mild treadmill exercise (9 m/min, 10% grade) increased heart rate (71 +/- 9.4 beats/min), mean arterial pressure (12 +/- 2.0 mmHg), and terminal aortic blood flow velocity (6 +/- 1.0 kHz). During exercise, a reduction of terminal aortic blood flow velocity (10.5 +/- 1.1%) reduced mixed venous PO2 18 +/- 6%. The gain of the muscle metaboreflex in the control condition was 14.6 +/- 2.9 mmHg/kHz. Efferent blockade reduced the gain 51 +/- 7%. However, combined cardiac efferent and afferent blockade increased the gain 207 +/- 64% above the efferent blocked condition and restored the gain to levels above those obtained in the control condition (18.3 +/- 4.6 mmHg/kHz). In addition, 15% blood volume expansion reduced the gain of the muscle metaboreflex regulation of mean arterial pressure and heart rate (44 +/- 9.5% and 41 +/- 12.0%, respectively). Thus cardiac afferents tonically inhibit the pressor response to a reduction in terminal aortic blood flow velocity during exercise.     

Presynaptic modulation of cholecystokinin release by protein kinase C in the rat hippocampus

The role of protein kinase C (PKC) in modulating the release of the octapeptide cholecystokinin (CCK-8) was investigated in rat hippocampal nerve terminals (synaptosomes). The PKC-activating phorbol ester 4beta-phorbol 12,13-dibutyrate (beta-PDBu) dose dependently (5-5,000 nM) increased CCK-8 release in a strictly Ca2+dependent way. This effect was observed only when synaptosomes were stimulated with the K+(A) channel blocker 4-aminopyridine (4-AP; 1 mM) but not with KCl (10-30 mM). The PDBu-induced exocytosis of CCK-8 was completely blocked by the two selective PKC inhibitors chelerythrine and calphostin-C and was not mimicked by alpha-PDBu, an inactive phorbol ester. In addition, an analogue of the endogenous PKC activator diacylglycerol, oleoylacetylglycerol, dose dependently increased CCK-8 exocytosis. Beta-PDBu (50-100 nM) also stimulated the 4-AP-evoked Ca2+-dependent release of the classic transmitter GABA, which co-localizes with CCK-8 in hippocampal interneurons. As a possible physiological trigger for PKC activation, the role of the metabotropic glutamate receptor was investigated. However, the broad receptor agonist (1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid did not stimulate, but instead inhibited, both the CCK-8 and the GABA exocytosis. In conclusion, presynaptic PKC may stimulate exocytosis of distinct types of co-localizing neurotransmitters via modulation of presynaptic K+ channels in rat hippocampus.