Electronmicroscopic study of human herpesvirus 6-infected human T cell lines superinfected with human immunodeficiency virus type 1

Melatonin release by chick cultured pineal cells increases during the dark periods and decreases during the light periods under light-dark cycles, and this rhythmic secretion is maintained under constant conditions with a period of almost 24 hr. The mechanisms by which the circadian oscillator drives the melatonin rhythm under constant conditions have not been elucidated enough. We examined the possibility that cyclic AMP-dependent protein kinase A is involved in the subjective nocturnal increase in melatonin release by chick pineal cells cultured under constant darkness. The subjective nocturnal increase of melatonin release was suppressed dose dependently by H8 (protein kinase inhibitor) and H89 (specific protein kinase A inhibitor), but not by calphostin C (specific protein kinase C inhibitor) in static cell cultures. In a cell perfusion experiment, 9 hr pulses of H8 and H89 starting at ZT 9 (CT 11.2) hr suppressed the subjective nocturnal increase in melatonin rhythm in dose-dependent manner without causing a phase shift. An intracellular Ca2+ chelator, O,O'-bis(2-aminophenoxy)ethyleneglycol-N,N,N',N'-tetraacetic acid tetraacetoxymethyl ester (BAPTA-AM), and extracellular Ca2+ chelators, O,O'-bis(2-aminophenoxy)ethyleneglycol-N,N,N',N'-tetraacetic acid tetrapotassium salt hydrate (BAPTA) and ethyleneglycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA), suppressed both the subjective nocturnal increases in melatonin release and cAMP levels dose dependently. This direct evidence strongly supports the hypothesis that cAMP-dependent protein kinase A may be involved in the subjective nocturnal increase in melatonin release by chick pineal cells and that intracellular Ca2+ plays an important role in pineal adenylate cyclase activation.     

Phase II study of irinotecan and etoposide in patients with metastatic non-small-cell lung cancer

Melatonin production in the chick pineal gland is high at night and low during the day. This rhythm reflects circadian changes in the activity of serotonin N-acetyltransferase (arylalkylamine N-acetyltransferase, AA-NAT; EC 2.3.1.87), the penultimate enzyme in melatonin synthesis. In contrast to the external regulation of pineal rhythms in mammals by the suprachiasmatic nucleus, rhythmic changes in AA-NAT activity in cultured chick pineal cells are controlled by an oscillator located in the pineal cells themselves. Here we present evidence that the chick pineal clock generates a rhythm in the abundance of AA-NAT mRNA in cultured cells that parallels the rhythm in AA-NAT activity. In contrast, elevating cAMP by forskolin treatment markedly increases AA-NAT activity without producing strong changes in AA-NAT mRNA levels, and lowering cAMP by norepinephrine treatment decreases enzyme activity without markedly decreasing mRNA. These results suggest that clock-controlled changes in AA-NAT activity occur primarily through changes at the mRNA level, whereas cAMP-controlled changes occur primarily through changes at the protein level. Related studies indicate that the clock-dependent nocturnal increase in AA-NAT mRNA requires gene expression but not de novo protein synthesis, and that AA-NAT mRNA levels are suppressed at all times of the day by a rapidly turning over protein. Further analysis of the regulation of chick pineal AA-NAT mRNA is likely to enhance our understanding of the molecular basis of vertebrate circadian rhythms.     

Neurotransmitter-induced novel modulation of a nonselective cation channel by a cAMP-dependent mechanism in rat pineal cells

In the rat, circadian rhythm in melatonin is regulated by noradrenergic and neuropeptide inputs to the pineal via adenosine 3',5'-cyclic monophosphate (cAMP)- and Ca2+-dependent mechanisms. We have identified a large conductance (170 pS), voltage-dependent, nonselective cation channel on rat pineal cells in culture that shows a novel mode of modulation by cAMP. Pituitary adenylate cyclase activating peptide (PACAP), norepinephrine, or 8-Br-cAMP increase channel open probability (Po) with a hyperpolarizing shift in voltage dependence such that the channel becomes active at resting membrane potentials. The increase in Po was accompanied by a change in current rectification properties such that the channel was transformed from being inactive at rest to an inwardly rectifying cation conductance in the presence of agonist, which depolarizes the cell. This channel is calcium insensitive, is blocked by Cs+, and shows a permeability sequence: K+ > Na+ >/= NH+4 > Li+. The data suggest that PACAP and norepinephrine acting through a cAMP-dependent mechanism modulate this nonselective cation channel, resulting in a slow onset depolarization that may be important in regulation of pineal cell excitability.     

Day/night differences in the stimulation of adenylate cyclase activity by calcium/calmodulin in chick pineal cell cultures: evidence for circadian regulation of cyclic AMP

In chick pineal cell culture, stimulation of adenylate cyclase with the diterpene forskolin was greater during the subjective night than during the subjective day. This rhythm of cyclic AMP (cAMP) stimulation mimicked the rhythm of unstimulated cAMP measured previously during LD cycles from flow-through culture. Direct measurement of adenylate cyclase activity in permeabilized cells revealed an adenylate cyclase activity activated by Ca2+/calmodulin during the night but not during the day. However, this difference in adenylate cyclase activity at two times of the circadian cycle is apparent only when permeabilized cells were prewashed with buffer containing GTE When cAMP was measured from flow-through cultures maintained in continuous darkness to determine whether a circadian clock may regulate cAMP, a low-amplitude rhythm was measured. The circadian rhythm of cAMP was similar to the cAMP rhythm previously measured on LD cycles except that the rhythm in darkness had a lower amplitude. Similar to the suppression of melatonin, cAMP was suppressed by light presented during the middle of the night. LD differences in nocturnal cAMP levels were abolished with dipyridamole, an inhibitor of cyclic GMP (cGMP) phosphodiesterase. These results suggest that the rhythm of cAMP in chick pineal cells involves the stimulation of adenylate cyclase by Ca2+/calmodulin during the night and a GTP-dependent suppression of adenylate cyclase activity during the day. The photic suppression of cAMP at night involves the activation of a dipyridamole-sensitive, cGMP phosphodiesterase.     

Effect of melatonin in the rat tail artery: role of K+ channels and endothelial factors

1. The role of endothelial factors and potassium channels in the action of the pineal hormone melatonin to potentiate vasoconstrictor responses was investigated in the isolated perfused tail artery of the rat. 2. Melatonin (100 nM) potentiated contractile responses to both adrenergic nerve stimulation and alpha1-adrenoceptor stimulation by phenylephrine. After removal of the endothelium, melatonin no longer caused potentiation. 3. The potentiating effect of melatonin was also lost when nitric oxide synthase was inhibited with L-NAME (10 nM). Thus potentiating effects depend on the presence of nitric oxide released by the endothelium. However, melatonin did not affect relaxation responses to acetylcholine in endothelium-intact arteries, nor did melatonin modulate relaxing responses to sodium nitroprusside in endothelium-denuded arteries. While melatonin does not appear to modulate agonist-induced release of nitric oxide nor its effect, melatonin may modulate nitric oxide production induced by flow and shear stress. 4. When the Ca2+-activated K+ channel opener, NS 1619 (10 microM), was present, potentiating effects of melatonin were restored in endothelium-denuded vessels. However, addition of the opener of ATP-sensitive K+ channels, cromakalim (3 microM), did not have the same restorative effect. Furthermore, addition of a blocker of Ca2+-activated K+ channels, tetraethylammonium (1 mM), significantly attenuated potentiating effects of melatonin. These findings support the hypothesis that melatonin inhibits the activity of large conductance Ca2+-activated K+ channels to produce its potentiating effects. 5. Thus in the rat perfused tail artery, potentiation of constriction by melatonin depends on the activity of both endothelial factors and Ca2+-activated K+ channels. Our findings suggest that melatonin inhibits endothelial K+ channels to decrease flow-induced release of nitric oxide as well as block smooth muscle K+ channels to enhance vascular tone.     

Signaltransduction in vascular endothelium: the role of intracellular calcium and ion channels

Endothelial cells (ECs) provide an ideal surface for blood flow. They inhibit the initiation of blood-clotting, but can also under certain conditions activate this process. ECs influence thrombolysis as well as thrombogenesis. They are "antigen-presenting cells" and play a key role in angiogenesis. In addition, ECs control the permeability of the barrier between bloodvessels and interstitium. One of their most important functions is the regulation of the diameter of the blood vessels and their adaptation to the demanded hemodynamic needs. The production and release of diverse compounds, which interfere with different neighboured target cells, initiate this plethora of functions. Ca2+ signals in endothelial cells play the key role in the release of NO, prostacyclin (PGI2), platelet activating factor (PAF), von Willebrand factor (vWF), tissue plasminogen activator (tPA) and tissue factor pathway inhibitor (TFPI). Changes in the intracellular Ca2+ concentration ([Ca2+]i) are determined by release from intracellular stores and entry through the plasma membrane. The diversity of Ca2+ entry pathways and mechanisms of their control are described. At least two different types of Ca2+ entry channels exist: 1. typical highly Ca2+ selective ion channels which might be activated by depletion of intracellular Ca2+ stores (Ca2+ release-activated Ca2+ channels, CRAC), and 2. Non-selective Ca2+ permeable cation channels (NSC). The latter shares many features with an NSC induced by expression of the protein TRPC3. These channels are only weakly operated by store depletion and require a permissive Ca2+ and Ins(1,4,5)P3 concentration in the cytosol. CRAC channels are possible indirectly involved in Ca2+ entry during mechano-stimulation of ECs. After activation of these entry channels, influx of Ca2+ depends on the driving force. The following ion channels play a pivotal role in regulation of the driving force for Ca2+ entry: an inwardly rectifying K+ channel, identified as Kir2.1, a large-conductance, Ca2+ activated K+ channel (hslo) and at least two Cl- channels (a volume regulated Cl- channel, VRAC, and a Ca2+ activated Cl- channel, CaCC). It will be explained how these ion channels interact in the regulation of the long-lasting (plateau-type) increase in [Ca2+]i which mainly controls NO-synthesis and release. Furthermore, it will be demonstrated that Ca2+ oscillations depend on intracellular events rather than Ca2+ entry from the extracellular space.     

What makes a mother? Interviews with women involved in egg donation and surrogacy

The fish pineal organ contains typical and, in some species, modified photoreceptor cells involved in the photoperiodic control of melatonin production. In the majority of species studied, the rhythm in melatonin production is driven by an intra-pineal circadian oscillator synchronized by the light:dark cycle. In the present study, it is shown that the endogenous rhythm in melatonin release of superfused pike pineals maintained under constant darkness is expressed at temperatures of 19 degrees C, 20 degrees C, 25 degrees C, and 30 degrees C (period > 24 hr), but not at temperatures of 10 degrees C and 15 degrees C. Under constant darkness, pineal fractions containing either typical photoreceptors, modified photoreceptors, or both behaved like total organs. Dissociated pike pineal cells, cultured statically at 20 degrees C, expressed a high amplitude rhythm in melatonin secretion under a light:dark cycle. Under constant darkness, circadian oscillations, which appeared better sustained than in organ culture, were also observed. This study provides the first evidence that the rhythmic production of melatonin, by a fish pineal, is driven by a population of circadian oscillators or clocks. It is hypothesized that each typical and modified photoreceptor might be the locus of a circadian clock. Damping of the overall rhythm under constant darkness might reflect the desynchronization (uncoupling) between these clocks and/or damping of individual oscillators.     

Differences in tryptophan binding to hepatic nuclei of NZBWF1 and Swiss mice: insight into mechanism of tryptophan''s effects

Catecholamine receptors of multiple classes have been shown to influence pineal melatonin synthesis in a species-specific manner. In these experiments, the effects of catecholamine receptor agonists on circadian melatonin rhythms of zebrafish (Danio rerio) pineal in vitro were examined. Cyclic application of adrenergic receptor agonists (norepinephrine, phenylephrine, clonidine, and isoproterenol) had no effect on zebrafish pineal melatonin release, nor on the circadian oscillator that regulates melatonin rhythms. Pineal melatonin release was partially suppressed by quinpirole, a D2 dopamine receptor agonist, but cyclic application of quinpirole did not reset the pineal circadian oscillator. Pineal melatonin release was unaffected by either dopamine or SKF38393, a D1 receptor agonist, suggesting that the effects of quinpirole were not mediated by dopamine receptors. The regulatory mechanisms underlying pineal melatonin rhythms appear to differ among teleosts.     

Regulation of heterologously expressed transient receptor potential-like channels by calcium ions

The Drosophila melanogaster gene product TRPL (transient receptor potential-like) is a Ca2+-permeable cation channel that contributes to the light-induced Ca2+ entry in Drosophila photoreceptors and bears homology to several recently cloned mammalian channels. Intracellular Ca2+ has been implicated to stimulate TRPL channels. This constitutes a potentially dangerous mechanism that may lead to Ca2+ overload. Therefore, we studied whether TRPL channels, like other Ca2+-permeable channels, are inhibited by intracellular Ca2+ concentrations in the micromolar range and whether this effect is mediated by calmodulin. In Sf9 cells expressing the TRPL gene along with histamine H1 receptors after infection with baculoviruses containing the corresponding complementary DNA, histamine-induced TRPL currents were inhibited by intracellular Ca2+ with an IC50 of 2.3 microM. Moreover, TRPL currents were reversibly attenuated by a preceding hyperpolarization. This attenuation reflected the action of an increased Ca2+ influx, since it was abolished in the absence of extracellular Ca2+ and enhanced by raising extracellular Ca2+ to 20 mM. Finally, the activity of TRPL channels in inside-out patches was reversibly inhibited by raising the Ca2+ concentration on the cytosolic side of the patches to 10-50 microM. Addition of calmodulin or the calmodulin inhibitor calmidazolium did not modify the inhibition of the TRPL by Ca2+. We conclude that high intracellular Ca2+ concentrations inhibit the TRPL, but no evidence was found for the requirement of calmodulin. This mechanism makes Ca2+ influx through the TRPL self-limiting. Furthermore, the TRPL may allow one to study the structural requirements for channel regulation by Ca2+.     

Melatonin, its receptors, and relationships with biological rhythm disorders

Melatonin is a neurohormone produced during the night by the pineal gland. Its secretion is regulated by circadian and seasonal variations in daylength, transmitted via visual projections to the suprachiasmatic nucleus which functions as a circadian clock in mammals. Melatonin has been proposed to act as an internal synchronizer of circadian rhythms generated at different levels of the organism. The chronobiotic effects of melatonin in humans have been mainly studied in circadian rhythm sleep disorders related to jet lag, shift work, blindness or aging. Alterations of the melatonin profiles have also been reported in other biological rhythm disorders.     

Cysteine string protein functions directly in regulated exocytosis

Cysteine string protein (Csp) is essential for neurotransmitter release in Drosophila. It has been suggested that Csp functions by regulating the activity of presynaptic Ca2+ channels, thus controlling exocytosis. We have examined the effect of overexpressing Csp1 in PC12 cells, a neuroendocrine cell line. PC12 cell clones overexpressing Csp1 did not show any changes in morphology, granule number or distribution, or in the levels of other key exocytotic proteins. This overexpression did not affect intracellular Ca2+ signals after depolarization, suggesting that Csp1 has no gross effect on Ca2+ channel activity in PC12 cells. In contrast, we show that Csp1 overexpression enhances the extent of exocytosis from permeabilized cells in response to Ca2+ or GTPgammaS in the absence of Ca2+. Because secretion from permeabilized cells is not influenced by Ca2+ channel activity, this represents the first demonstration that Csp has a direct role in regulated exocytosis.     

Voltage-dependent Ca2+ channels in arterial smooth muscle cells

The past years have seen some significant advances in our understanding of the functional and molecular properties of voltage-dependent Ca2+ channels in arterial smooth muscle. Molecular cloning and expression studies together with experiments on native voltage-dependent Ca2+ channels revealed that these channels are built upon a molecular structure with properties appropriate to function as the main source for Ca2+ entry into arterial smooth muscle cells. This Ca2+ entry regulates intracellular free Ca2+, and thereby arterial tone. We summarize several avenues of recent research that should provide significant insights into the functioning of voltage-dependent Ca2+ channels under conditions that occur in arterial smooth muscle. These experiments have identified important features of voltage-dependent Ca2+ channels, including the steep steady-state voltage-dependence of the channel open probability at steady physiological membrane potentials between -60 and -30 mV, and a relatively high permeation rate at physiological Ca2+ concentrations, being about one million Ca2+ ions/s at -50 mV. This calcium permeation rate seems to be a feature of the pore-forming Ca2+ channel alpha1 subunit, since it was identical for native channels and the expressed alpha1 subunit alone. The channel activity is regulated by dihydropyridines, vasoactive hormones and intracellular signaling pathways. While the membrane potential of smooth muscle cells primarily regulates arterial muscle tone through alterations in Ca2+ influx through dihydropyridine-sensitive voltage-dependent ('L-type') Ca2+ channels, the role of these channels in the differentiation and proliferation of vascular smooth muscle cells is less clear. We discuss recent findings suggesting that other Ca2+ permeable ion channels might be important for the control of Ca2+ influx in dedifferentiated vascular smooth muscle cells.     

Sigma receptor modulation of noradrenergic-stimulated pineal melatonin biosynthesis in rats

Because sigma receptors are richly concentrated in the rat pineal gland, the present study was performed to investigate their possible role in the modulation of melatonin production. To this purpose, we assessed in vivo the effects of the sigma-receptor ligands 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine on the rat pineal gland activity during either the daytime or the nighttime. Compared with vehicle, 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine potentiated the enhancement of N-acetyltransferase activity and pineal melatonin content induced by isoproterenol administration during the daytime, whereas they did not affect the diurnal basal biosynthetic activity of the gland. Conversely, at night, 1,3-di(2-tolyl)guanidine and (+)-N-allylnormetazocine enhanced significantly the physiological increases in both pineal N-acetyltransferase activity and melatonin levels. This enhancement was prevented by pretreatment with rimcazole, a specific sigma-receptor antagonist. These findings suggest that, in rats, the activation of pineal sigma-receptor sites does not affect the biosynthetic activity of the pineal gland during daytime, whereas it potentiates the production of melatonin when the gland is noradrenergically stimulated either by isoproterenol administration or by the endogenously released norepinephrine at nighttime.     

Localized secretion of ATP and opioids revealed through single Ca2+ channel modulation in bovine chromaffin cells

In bovine chromaffin cells, the Ca2+ channels involved in exocytosis are effectively inhibited by ATP and opioids that are coreleased with catecholamines during cell activity. This autocrine loop causes a delay in Ca2+ channel activation that is quickly removed by preceding depolarizations. Changes in Ca2+ channel gating by secreted products thus make it possible to correlate Ca2+ channel activity to secretory events. Here, using cell-attached patch recordings, we found a remarkable correlation between delayed Ca2+ channel openings and neurotransmitter secretion induced by either local or whole-cell Ba2+ stimulation. The action is specific for N- and P/Q-type channels and largely prevented by PTX and mixtures of purinergic and opioid receptor antagonists. Overall, our data provide evidence that exocytosis, viewed through the autocrine inhibition of non-L-type channels, is detectable in membrane patches of approximately 1 microm2 distributed over 30%-40% of the total cell surface, while Ca2+ channels and autoreceptors are uniformly distributed over most of the cell membrane.     

Regulation of the intracellular concentration of free calcium ions in pinealocytes of the rainbow trout and the rat

Together with cAMP, calcium ions play an important role in the regulation of melatonin synthesis in the pineal organ of all vertebrate species, irrespective of the conspicuous phylogenetic transformation of the melatonin-producing cell, the pinealocyte. Here we address the question how the intracellular concentration of free calcium ions [Ca2+]i is regulated in directly light-sensitive trout pinealocytes and in rat pinealocytes which have lost the direct light sensitivity and respond to norepinephrine. Isolated pinealocytes identified by the S-antigen immunoreaction were investigated by means of the fura-2 technique, image analysis and patch clamp recordings. Approximately 30% of the trout pinealocytes exhibited spontaneous [Ca2+]i oscillations that were not affected by light or dark adaptation of the cells. Removal of extracellular Ca2+ or application of 10 microM nifedipine caused a reversible breakdown of the [Ca2+]i oscillations. Treatments with 60 mM KCl and nifedipine suggest that voltage-gated L-type calcium channels play a major role in the regulation of [Ca2+]i in both oscillating and nonoscillating trout pinealocytes. Experiments with thapsigargin (2 microM) revealed the presence of intracellular calcium stores in 80% of the trout pinealocytes, but their role in the regulation of [Ca2+]i remains elusive. Norepinephrine had no apparent effect on [Ca2+]i in any trout pinealocyte. In rat pinealocytes, [Ca2+]i did not show spontaneous oscillations. Norepinephrine evoked a dramatic biphasic rise in [Ca2+]i in more than 95% of the cells via stimulation of alpha1-adrenergic receptors. The response reflects a combination of calcium mobilization from intracellular, thapsigargin-sensitive calcium stores and an increased calcium influx. Voltage-gated calcium channels of the L-type are present in the rat pinealocyte membrane, but they are not involved in the norepinephrine-induced calcium response. These channels, however, mediate the increase in calcium influx which is observed in virtually all rat pinealocytes upon stimulation with acetylcholine or nicotine. The results show that the mechanisms which regulate [Ca2+]i in pinealocytes are complex and differ considerably between poikilothermic and mammalian species.     

Properties of a novel pH-dependent Ca2+ permeation pathway present in male germ cells with possible roles in spermatogenesis and mature sperm function

Rises of intracellular Ca2+ ([Ca2+]i) are key signals for cell division, differentiation, and maturation. Similarly, they are likely to be important for the unique processes of meiosis and spermatogenesis, carried out exclusively by male germ cells. In addition, elevations of [Ca2+]i and intracellular pH (pHi) in mature sperm trigger at least two events obligatory for fertilization: capacitation and acrosome reaction. Evidence implicates the activity of Ca2+ channels modulated by pHi in the origin of these Ca2+ elevations, but their nature remains unexplored, in part because work in individual spermatozoa are hampered by formidable experimental difficulties. Recently, late spermatogenic cells have emerged as a model system for studying aspects relevant for sperm physiology, such as plasmalemmal ion fluxes. Here we describe the first study on the influence of controlled intracellular alkalinization on [Ca2+]i on identified spermatogenic cells from mouse adult testes. In BCECF [(2',7')-bis(carboxymethyl)- (5, 6)-carboxyfluorescein]-AM-loaded spermatogenic cells, a brief (30-60 s) application of 25 mM NH4Cl increased pHi by approximately 1.3 U from a resting pHi approximately 6.65. A steady pHi plateau was maintained during NH4Cl application, with little or no rebound acidification. In fura-2-AM-loaded cells, alkalinization induced a biphasic response composed of an initial [Ca2+]i drop followed by a two- to threefold rise. Maneuvers that inhibit either Ca2+ influx or intracellular Ca2+ release demonstrated that the majority of the Ca2+ rise results from plasma membrane Ca2+ influx, although a small component likely to result from intracellular Ca2+ release was occasionally observed. Ca2+ transients potentiated with repeated NH4Cl applications, gradually obliterating the initial [Ca2+]i drop. The pH-sensitive Ca2+ permeation pathway allows the passage of other divalents (Sr2+, Ba2+, and Mn2+) and is blocked by inorganic Ca2+ channel blockers (Ni2+ and Cd2+), but not by the organic blocker nifedipine. The magnitude of these Ca2+ transients increased as maturation advanced, with the largest responses being recorded in testicular sperm. By extrapolation, these findings suggest that the pH-dependent Ca2+ influx pathway could play significant roles in mature sperm physiology. Its pharmacology and ion selectivity suggests that it corresponds to an ion channel different from the voltage-gated T-type Ca2+ channel also present in spermatogenic cells. We postulate that the Ca2+ permeation pathway regulated by pHi, if present in mature sperm, may be responsible for the dihydropyridine-insensitive Ca2+ influx required for initiating the acrosome reaction and perhaps other important sperm functions.     

Stimulation of cyclic ADP-ribose synthesis by acetylcholine and its role in catecholamine release in bovine adrenal chromaffin cells

Cyclic ADP-ribose (cADPR) is suggested to be a novel messenger of ryanodine receptors in various cellular systems. However, the regulation of its synthesis in response to cell stimulation and its functional roles are still unclear. We examined the physiological relevance of cADPR to the messenger role in stimulation-secretion coupling in cultured bovine adrenal chromaffin cells. Sensitization of Ca2+-induced Ca2+ release (CICR) and stimulation of catecholamine release by cADPR in permeabilized cells were demonstrated along with the contribution of CICR to intracellular Ca2+ dynamics and secretory response during stimulation of intact chromaffin cells. ADP-ribosyl cyclase was activated in the membrane preparation from chromaffin cells stimulated with acetylcholine (ACh), excess KCl depolarization, and 8-bromo-cyclic-AMP. ACh-induced activation of ADP-ribosyl cyclase was dependent on the influx of Ca2+ into cells and on the activation of cyclic AMP-dependent protein kinase. These and previous findings that ACh activates adenylate cyclase by Ca2+ influx in chromaffin cells suggested that ACh induces activation of ADP-ribosyl cyclase through Ca2+ influx and cyclic AMP-mediated pathways. These results provide evidence that the synthesis of cADPR is regulated by cell stimulation, and the cADPR/CICR pathway forms a significant signal transduction for secretion.     

Contrasting biophysical and pharmacological properties of T-type and R-type calcium channels

In contrast to other kinds of voltage-gated Ca2+ channels, the underlying molecular basis of T-type and R-type channels is not well-understood. To facilitate comparisons with cloned Ca2+ channel subunits, we have carried out a systematic analysis of the properties of T-type currents in undifferentiated NG108-15 cells and R-type currents in cerebellar granule neurons. Marked differences were found in their biophysical and pharmacological features under identical recording conditions. T-type channels became activated at potentials approximately 25 mV more negative than R-type channels; however, T-type channels required potentials approximately 15 mV less negative than R-type channels to be available. Accordingly, T-type channels display a much larger overlap between the curves describing inactivation and activation, making them more suitable for generating sustained Ca2+ entry in support of secretion or pacemaker activity. In contrast, R-type channels are not equipped to provide a steady current, but are very capable of supplying transient surges of Ca2+ influx. In response to a series of increasingly strong depolarizations T-type and R-type Ca2+ channels gave rise to very different kinetic patterns. T-type current records crossed each other in a characteristic pattern not found for R-type currents. These biophysical distinctions were independent of absolute membrane potential and were, therefore, complementary to the conventional categorization of T- and R-type Ca2+ channels as low- and high-voltage activated. R-type channels deactivated approximately eight-fold more quickly than T-type channels, with clear consequences for the generation of divalent cation influx during simulated action potentials. Pharmacological comparisons revealed additional contrasts. R-type current was responsive to block by omega-Aga IIIA but not nimodipine, while the opposite was true for T-type current. Both channel types were potently inhibited by the non-dihydropyridine compound mibefradil. In all respects examined, R-type currents were similar to currents derived from expression of the alpha1E subunit whereas T-type currents were not.