Xerostomia and pathological thirst are troublesome complications of diabetes mellitus associated with impaired functioning of salivary glands; however, their cellular mechanisms are not yet determined. Isolated acinar cells were loaded with Ca(2+) indicators fura-2/AM for measuring cytosolic Ca(2+) concentration ([Ca(2+)](i)) or mag-fura-2/AM-inside the endoplasmic reticulum (ER). We found a dramatic decrease in pilocarpine-stimulated saliva flow, protein content and amylase activity in rats after 6 weeks of diabetes vs. healthy animals. This was accompanied with rise in resting [Ca(2+)](i) and increased potency of acetylcholine (ACh) and carbachol (CCh) but not norepinephrine (NE) to induce [Ca(2+)](i) transients in acinar cells from diabetic animals. However,[Ca(2+)](i) transients mediated by Ca(2+) release from ER stores (induced by application of either ACh, CCh, NE, or ionomycin in Ca(2+)-free extracellular medium) were decreased under diabetes. Application of inositol-1,4,5-trisphosphate led to smaller Ca(2+) release from ER under the diabetes. Both plasmalemma and ER Ca(2+)-ATPases activity was reduced and the latter showed the increased affinity to ATP under the diabetes. We conclude that the diabetes caused impairment of salivary cells functions that, on the cellular level, associates with Ca(2+) overload, increased Ca(2+)-mobilizing ability of muscarinic but not adrenergic receptors, decreased Ca(2+)-ATPases activity and ER Ca(2+) content.

The influence of extracellular pH (pH(o)) on low-voltage-activated calcium channels of acutely isolated DRG neurons of rats was examined using the whole cell patch-clamp technique. It has been found that in the neurons of middle size with capacitance C=60+/-4.8 pF (mean+/-S.E., n=8) extracellular acidification from pH(o) 7.35 to pH(o) 6.0 significantly and reversibly decreased LVA calcium current densities by 75+/-3.7%, shifted potential for half-maximal activation to more positive voltages by 18.7+/-0.6 mV with significant reduction of its voltage dependence. The half-maximal potential of steady-state inactivation shifted to more positive voltages by 12.1+/-1.7 mV (n=8) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of middle size have midpoint pK(a)=6.6+/-0.02 and hill coefficient h=0.94+/-0.04 (n=5). In small cells with capacitance C=26+/-3.6 pF (n=5), acidosis decreased LVA calcium current densities only by 15.3+/-1.3% and shifted potential for half-maximal activation by 5.5+/-1.0 mV with reduction of its voltage dependence. Half-maximal potential of steady-state inactivation shifted to more positive voltages by 10+/-1.6 mV (n=4) and also became less voltage dependent. Dose-response curves for the dependence of maximum values of LVA currents on external pH in neurons of small size have midpoint pK(a)=7.9+/-0.04 and hill coefficient h=0.25+/-0.1 (n=4). These two identified types of LVA currents besides different pH sensitivity demonstrated different kinetic properties. The deactivation of LVA currents with weak pH sensitivity after switching off depolarization to -30 mV had substantially longer decay time than do currents with strong pH sensitivity (tau(d) approximately 5 ms vs. 2 ms respectively). It was found that the prolongation of depolarization steps slows the subsequent deactivation of T-type currents in small DRG neurons. Deactivation traces in these neurons were better described by the sum of two exponentials. Thus, we suppose that T-type channels in small DRG neurons are presented mostly by alpha1I subunit. We suggest that these two types of LVA calcium channels with different sensitivity to external pH can be differently involved in the origin of neuropathic changes.

Adrenal chromaffin cells secrete catecholamines in response to cholinergic receptor activation by acetylcholine (ACh). Characteristics of Ca(2+) transients induced by activation of nicotinic (nAChRs) and muscarinic (mAChRs) receptors were analyzed using Fura-2 fluorescent measurements on rat chromaffin cells. We first found two populations of chromaffin cells, which differently responded on AChR stimulation. In the first group ([Formula: see text]-cells), consecutive ACh applications evoked persistent Ca(2+) transients, whereas desensitizing transients were observed in the other group ([Formula: see text]-cells). The AChR agonists and antagonists precisely imitated or abolished the ACh action on [Formula: see text]- and [Formula: see text]-type cells, respectively. Cytochemical staining showed that [Formula: see text]-cells contained adrenaline, whereas [Formula: see text]-cells-noradrenaline. Thus, for the first time we found that nAChRs and mAChRs are differentially expressed in adrenergic and noradrenergic chromaffin cells, respectively. Our data suppose that chromaffin cells can be differentially regulated by incoming ACh signals and in such way release different substances-adrenaline and noradrenaline.

In view of the present controversy related to the potential beneficial effects of clinical dehydroepiandrosterone (DHEA) treatments, and considering our own previous results that reveal an influence of this steroid in pituitary hyperplasia development in vivo in rats, we decided to evaluate the role of DHEA in prolactin and GH secretion, as well as in second messengers involved, in cultured rat anterior pituitary cells. DHEA (1 x 10(-5) to 1 x 10(-7) M) did not modify basal GH or prolactin release, and a prolactin inhibitory effect was observed only for androstenediol, a metabolite of DHEA. DHEA partially prevented dopamine (1 x 10(-6) M)-induced prolactin inhibition and facilitated the prolactin-releasing effect of 10(-8) M Ang II, without modifying the resulting Ca2+(i) mobilization. Furthermore, DHEA potentiated the GH release and cAMP production induced by 1 x 10(-8) M GHRH. Finally, DHEA partially reversed the inhibitory effect of 1 x 10(-8) M somatostatin on GH, but not prolactin, release. We conclude that DHEA in vitro, directly or indirectly through conversion into metabolites, is able to modulate the hormonal response of the pituitary to hypothalamic regulators. It can enhance pituitary prolactin release and induce GH secretion. These effects could help explain some of the side effects observed in prolonged DHEA treatments in vivo and should be taken into account when considering its use in human clinical trials.

Cortisol was previously shown to rapidly (10-20 min) reduce the release of prolactin (PRL) from pituitary glands of tilapia (Oreochromis mossambicus). This inhibition of PRL release by cortisol is accompanied by rapid reductions in (45)Ca(2+) and cAMP accumulation. Cortisol's early actions occur through a protein synthesis-independent pathway and are mimicked by a membrane-impermeable analog. The signaling pathway that mediates rapid, nongenomic membrane effects of glucocorticoids is poorly understood. Using the advantageous characteristics of the teleost pituitary gland from which a nearly pure population of PRL cells can be isolated and incubated in defined medium, we examined whether cortisol rapidly reduces intracellular free calcium (Ca(i)(2+)) and suppresses L-type voltage-gated ion channel activity in events that lead to reduced PRL release. Microspectrofluorometry, used in combination with the Ca(2+)-sensitive dye fura 2 revealed that cortisol reversibly reduces basal and hyposmotically induced Ca(i)(2+) within seconds (P < 0.001) in dispersed pituitary cells. Somatostatin, a peptide known to inhibit PRL release through a membrane receptor-coupled mechanism, similarly reduces Ca(i)(2+). Under depolarizing [K(+)], the L-type calcium channel agonist BAY K 8644, a factor known to delay the closing of L-type Ca(2+) channels, stimulates PRL release in a concentration-dependent fashion (P < 0.01). Cortisol (and somatostatin) blocks BAY K 8644-induced PRL release (P < 0.01; 30 min), well within the time course over which its actions occur, independent of protein synthesis and at the level of the plasma membrane. Results indicate that cortisol inhibits tilapia PRL release through rapid reductions in Ca(i)(2+) that likely involve an attenuation of Ca(2+) entry through L-type voltage-gated Ca(2+) channels. These results provide further evidence that glucocorticoids rapidly modulate hormone secretion via a membrane-associated mechanism similar to that observed with the fast effects of peptides and neurotransmitters.

Orexins, orexigenic neuropeptides, have recently been discovered in lateral hypothalamus and play an important role in the regulation of pituitary hormone secretion. Two subtypes of orexin receptors (orexin-1 and orexin-2) have been demonstrated in pituitaries. In this experiment, the effects of orexins on voltage-gated Ca2+ currents and the GH release in primary cultured ovine somatotropes were examined. Voltage-gated Ca2+ currents were isolated in ovine somatotropes as L, T, and N currents using whole-cell patch-clamp techniques and specific Ca2+ channel blocker and toxin. Application of orexin-A or orexin-B (100 nM) significantly, dose-dependently, and reversibly increased only nifedipine-sensitive L-type Ca2+ current. Inhibitors of PKC (calphostin C, PKC inhibitory peptide) but not inhibitors of PKA (H89, PKA inhibitory peptide) cancelled the increase in the L current by orexins. Co-administration of orexin-A and GHRH (10 nM) showed an additive effect on the L current. Specific intracellular Ca2+-store-depleting reagent, thapsigargin (1 microM), did not affect the orexin-induced increase in the L current. Orexin-B alone slightly increased GH release and co-administration of orexin-A and GHRH synergistically stimulated GH secretion in vitro. It is therefore suggested that orexins may play an important role in regulating GHRH-stimulated GH secretion through an increase in the L-type Ca2+ current and the PKC-mediated signaling pathways in ovine somatotropes.

Voltage-operated calcium channels play a crucial role in signal transduction in many excitable and non-excitable cell types. While a rapid modulation of their activity by hormone-activated kinases and/or G proteins has been recognized for a long time, a sustained control of their expression level has been only recently demonstrated. In adrenal H295R cells, for example, aldosterone treatment selectively increased low threshold T-type calcium current density without affecting L-type currents. Antagonizing the mineralocorticoid receptor (MR) with spironolactone prevented aldosterone action on T-type currents. By RT-PCR, we detected in these cells the presence of two different isoforms of L-type channels, alpha(1)C and alpha(1)D, and one isoform of T channel, alpha(1)H. A second T channel isoform (alpha(1)G) was also observed under particular culture conditions. Quantification of the specific messenger RNA by real time RT-PCR allowed us to show a 40% increase of the alpha1H messenger levels upon aldosterone treatment (alpha(1)G was insensitive), a response that was also completely prevented by spironolactone. Because T-type, but not L-type channel activity is linked to steroidogenesis, this modulation represents a positive, intracrine feed back mechanism exerted by aldosterone on its own production.Aldosterone has been also implicated in the pathogenesis and progression of ventricular hypertrophy and heart failure independently of its action on arterial blood pressure. We have observed that, in rat neonatal cardiomyocytes, aldosterone increases (by two-fold) L-type calcium current amplitude in ventricular but not in atrial cells. No significant effect of aldosterone could be detected on T-type currents, that were much smaller than L-type currents in these cells. However, aldosterone exerted opposite effects on T channel isoform expression, increasing alpha(1)H and decreasing alpha(1)G. Although the functional role of T channels is still poorly defined in ventricular cardiomyocytes, an overexpression of alpha(1)H could be partially responsible for the arrhythmias linked to hyperaldosteronism.Finally, T channels also appear to be involved in the neuroendocrine differentiation of prostate epithelial cells, a poor prognosis in prostate cancer. We have shown that the only calcium channel expressed in the prostatic LNCaP cells is the alpha(1)H isoform and that induction of cell differentiation with cAMP leads to a concomitant increase in both T-type current and alpha(1)H mRNA. In spite of the presence of MR in these cells, aldosterone only modestly increased alpha(1)H mRNA levels. A functional role for these channels was suggested by the observation that low nickel concentrations prevent neuritic process outgrowth.In conclusion, it appears that T-type calcium channel expression vary in different patho-physiological conditions and that aldosterone, in several cell types, is able to modulate this expression.

Decrease in extracellular osmolarity ([Os]e) results in stimulation of hormone secretion from pituitary cells. Different mechanisms can account for this stimulation of hormone secretion. In this study we examined the possibility that hyposmolarity directly modulates voltage-gated calcium influx in pituitary cells. The effects of hyposmolarity on L-type (IL) and T-type (IT) calcium currents in pituitary cells were investigated by using two hyposmotic stimuli, moderate (18-22% decrease in [Os]e) and strong (31-32% decrease in [Os]e). Exposure to moderate hyposmotic stimuli resulted in three response types in IL (a decrease, a biphasic effect, and an increase in IL) and in increase in IT. Exposure to strong hyposmotic stimuli resulted only in increases in both IL and IT. Similarly, in intact pituitary cells (perforated patch method), exposure to either moderate or strong hyposmotic stimuli resulted only in increases in both IL and IT. Thus it appears that the main effect of decrease in [Os]e is increase in calcium channel currents. This increase was differential (IL were more sensitive than IT) and voltage independent. In addition, we show that these hyposmotic effects cannot be explained by activation of an anionic conductance or by an increase in cell membrane surface area. In conclusion, this study shows that hyposmotic swelling of pituitary cells can directly modulate voltage-gated calcium influx. This hyposmotic modulation of IL and IT may contribute to the previously reported hyposmotic stimulation of hormone secretion. The mechanisms underlying these hyposmotic effects and their possible physiological relevance are discussed.

Hair cell calcium channels regulate membrane excitability and control synaptic transmission. The present investigations focused on determining whether calcium channels vary between hair cells of different characteristic frequencies or if multiple channel types exist within a hair cell, each serving a different function. To this end, turtle auditory hair cells from high-(317 +/- 27 Hz) and low-frequency (115 +/- 6 Hz) positions were voltage clamped using the whole-cell recording technique, and calcium currents were characterized based on activation, inactivation and pharmacological properties. Pharmacological sensitivity to dihydropyridines (nimodipine, Bay K 8644), benzothiazepines (diltiazem) and acetonitrile derivatives (verapamil, D600) and the insensitivity to non-L-type calcium channel antagonists support the conclusion that only L-type calcium channels were present. Fast activation rise times (< 0.5 ms), hyperpolarized half-activation potentials and a relative insensitivity to nimodipine suggest the channels were of the alpha1D (CaV1.3) variety. Although no pharmacological differences were found between calcium currents obtained from high- and low-frequency cells, low-frequency cells activated slightly faster and at hyperpolarized potentials, with half-activating voltages of -43 +/- 1 mV compared to -35 +/- 1 mV. Inactivation was observed in both high- and low-frequency cells. The time course of inactivation required three time constants for a fit. Long depolarizations could result in complete inactivation. The voltage of half-inactivation was -40 +/- 2 mV for high-frequency cells and -46 +/- 2 mV for low-frequency cells. Calcium channel inactivation did not significantly alter hair cell electrical resonant properties elicited from protocols where the membrane potential was hyperpolarized or depolarized prior to characterizing the resonance. A bell-shaped voltage dependence and modest sensitivities to intracellular calcium chelators and external barium ions suggest that inactivation was calcium dependent.

Orexins, orexigenic neuropeptides, are secreted from lateral hypothalamus and orexin receptors are expressed in the pituitary. Since growth hormone (GH) secreted from pituitary is integrally linked to energy homeostasis and metabolism, we studied the effect of orexin-B on voltage-gated Ca(2+) currents and the related signalling mechanisms in primary cultured ovine somatotropes using whole-cell patch-clamp techniques. With a bath solution containing TEA-Cl (40 mM) and Tetrodotoxin (TTX)(1 microM), three subtypes of Ca(2+) currents, namely the long-lasting (L), transient (T), and N currents, were isolated using different holding potentials (-80 and -30 mV) in combination with specific Ca(2+) channel blockers (nifedipine and omega-conotoxin). About 75% of the total current amplitude was contributed by the L current, whereas the N and T currents accounted for the rest. Orexin-B (1-100 nM) dose-dependently and reversibly increased only the L current up to approximately 125% of the control value within 4-5 min. Neither a specific protein kinase A (PKA) blocker (H89, 1 microM) nor an inhibitory peptide (PKI, 10 microM) had any effect on the increase in L current by orexin-B. The orexin-B-induced increase in the L current was abolished by concurrent treatment with calphostin C (Cal-C, 100 nM), protein kinase C (PKC) inhibitory peptide (PKC(19-36), 1 microM), or by pretreatment with phorbol-12,13-dibutyrate (PDBu)(0.5 microM) for 16 h (a downregulator of PKC). Orexin-B also increased in vitro GH secretion in a dose-dependent manner. We conclude that orexin-B increases the L-type Ca(2+) current and GH secretion through orexin receptors and PKC-mediated signalling pathways in ovine somatotropes.

The effect of chronic dexamethasone (DEX) treatment (4-5 days) on Na(+) channel expression was examined in a clonal strain of rat pituitary cells secreting growth hormone (GH) and prolactin (GH3 cells). Using whole-cell patch clamp recording, we found that DEX (1 microM) induces an 80% decrease in Na(+) current density. No concomitant changes in current kinetics or voltage dependence of Na(+) channel function were detected. Instead, the decrease in current density was accompanied by a similar reduction in maximal Na(+) conductance, suggesting the loss of Na(+) channels from the plasma membrane. Accordingly, saxitoxin binding assays carried out on intact cells showed that the average number of Na(+) channels per cell is markedly decreased by DEX. Thus, this glucocorticoid inhibits the cell surface expression of Na(+) channels when chronically applied to GH3 cells.

Orexins are recently discovered neuropeptides that play an important role in the regulation of hormone secretion, and their receptors have been recently demonstrated in the pituitary. The effects of orexin-A on voltage-gated Ca2+ currents and GH release in primary cultured ovine somatotropes were examined. The expression of orexin-1 receptor was demonstrated by RT-PCR in ovine somatotropes, from which Ca2+ currents were also isolated as L, T, and N currents. Application of orexin-A (100 nM) significantly and reversibly increased only the L current, and coadministration of orexin-A and GHRH (10 nM) showed an additive effect on this current, but no effect of orexin-A was observed on either T or N current. Furthermore, the orexin-A-induced increase in the L current was completely abolished by the inhibition of protein kinase C (PKC) activity using calphostin C (100 nM), phorbal 12,13-dibutyrate pretreatment (0.5 micro M) for 16 h or specific PKC inhibitory peptide PKC(19-36)(1 mM). However, the increase in L current by orexin-A was sustained when cells were preincubated with a specific protein kinase A blocker H89 (1 micro M) or a specific intracellular Ca2+ store depleting reagent thapsigargin (1 micro M). Finally, orexin-A alone did not significantly increase GH release, but coadministration of orexin-A and GHRH showed a synergistic effect on GH secretion in vitro. Our results therefore suggest that orexin-A may play an important role in regulating GHRH-stimulated GH secretion through the enhancement of the L-type Ca2+ current and the PKC-mediated signaling pathway in ovine somatotropes.

Long-term in vivo treatment with synthetic GH-releasing peptides (GHRPs) enhances the release of GH induced by endogenous GHRH. The mechanism for such an enhancement on GH release is unknown. In this experiment, somatotropes were obtained from ovine pituitaries by enzyme dissociation and enriched by density centrifugation. Membrane voltage and currents were recorded with whole-cell patch-clamp configuration. After 48-h treatment with GHRP-2 (10(-8) M), the percentage of cells with spontaneous action potential was increased (51 vs. 27%) without change of resting potential. This GHRP-2 treatment also increased the amplitude of voltage-gated K+ currents (predominantly transient A-type-like current but also delayed rectifier or K-type-like current) without modification of biophysical kinetics. Down-regulation of protein kinase C (PKC) with phorbol 12-myristate 13-acetate at the time of adding GHRP-2 blocked the increase in K+ currents. Inclusion of calphostin C (PKC inhibitor) but not H(89)(protein kinase A inhibitor) significantly reduced the increase in K+ currents by GHRP-2. Inclusion of actinomycin D (transcription inhibitor) or cycloheximide (protein synthesis inhibitor) abolished the increase in K+ currents. These data indicate that 48-h GHRP-2 treatment increases the density of K+ channels via PKC and channel protein synthesis. Such a modification on K+ channels by GHRP-2 may be partially responsible for the change of somatotrope electrophysiological properties and sensitivity to GHRH stimulation.