Ca2+ signalling and Ca2+-activated ion channels in exocrine acinar cells

The development of the calcium signalling field, from its early beginnings some 40 years ago to the present, is described. Calcium signalling in exocrine gland acinar cells and the effects of neurotransmitter- or hormone-elicited rises in the cytosolic calcium ion concentration on ion channel gating are reviewed. The highly polarized arrangement of the organelle systems in living acinar cells is described as well as its importance for the physiologically relevant local and polarized calcium signalling events.     

The effects of fatty acids on phosphoinositide synthesis and myo-inositol accumulation in exocrine pancreas

The effects of arachidonic acid (20:4) on phosphoinositide turnover were examined in rat pancreatic acinar cells prelabeled with myo-[3H]inositol. Arachidonic acid (50 microM) increased the accumulation of myo-[3H]inositol, but not that of [3H]inositol monophosphate, [3H]inositol bisphosphate, or [3H]inositol trisphosphate. By contrast, 10 microM carbamoylcholine increased the accumulation of all four compounds. A combination of arachidonic acid plus carbamoylcholine caused a selective and marked accumulation of myo-[3H]inositol, which was abolished by 10 mM LiCl. Arachidonic acid (10-100 microM) produced a concentration-dependent inhibition of myo-[3H]inositol incorporation into phosphoinositides and markedly depressed carbamoylcholine-induced increases in myo-[3H]inositol incorporation into inositol phospholipids. Several other unsaturated and saturated fatty acids failed to elicit a synergistic response with carbamoylcholine in stimulating myo-[3H]inositol accumulation and did not retard the incorporation of myo-[3H]inositol into phosphoinositides. The fact that eicosapentaenoic acid (20:5), but not arachidic acid (20:0), mimicked the depressant effect of arachidonate on phosphoinositide labeling suggests that the degree of unsaturation of the fatty acid, rather than chain length, is important for inhibition of phosphoinositide synthesis. The arachidonate-induced decrease in myo-[3H]inositol incorporation was accompanied by a reduction in the steady state level of [32P]phosphatidylinositol 4,5-bisphosphate. The mass of arachidonic acid liberated in response to carbamoylcholine was measured by gas chromatography-mass spectrometry, and the time course of stimulated arachidonate accumulation paralleled that of inositol phosphate accumulation and amylase release. These observations suggest that in exocrine pancreas, endogenous arachidonic acid serves as a negative feedback regulator of phosphoinositide turnover.     

Combined phosphoinositide and Ca2+ signals mediating receptor specificity toward neuronal Ca2+ channels

Phosphatidylinositol 4,5-bisphosphate (PIP(2)) regulates Ca(2+) (I(Ca)) and M-type K(+) currents in superior cervical ganglion sympathetic neurons. In those cells, M(1) muscarinic and AT(1) angiotensin types do not elicit Ca(2+)(i) signals and suppress both currents via depletion of PIP(2), whereas the B(2) bradykinin and P2Y purinergic types elicit robust IP(3)-mediated [Ca(2+)](i) rises and neither deplete PIP(2) nor inhibit I(Ca). We have suggested that this specificity arises from differential Ca(2+)(i) signals underlying receptor-specific stimulation of PIP(2) synthesis by phosphatidylinositol (PI) 4-kinase. Here, we investigate which PI 4-kinase isoform underlies this signal, whether stimulation of PI 4-phosphate 5-kinase is also required, and the origin of receptor-specific Ca(2+)(i) signals. Recordings of I(Ca) were used as a PIP(2) "biosensor." In control, stimulation of M(1), but not B(2) or P2Y, receptors robustly suppressed I(Ca). However, when PI 4-kinase IIIβ, diacylglycerol kinase, Rho, or Rho-kinase was blocked, agonists of all three receptors robustly suppressed I(Ca). Overexpression of exogenous M(1) receptors yielded large [Ca(2+)](i) rises by muscarinic agonist, and transfection of wild-type IRBIT decreased Ca(2+)(i) signals, whereas dominant negative IRBIT-S68A had little effect on B(2) or P2Y responses but greatly increased muscarinic responses. We conclude that overlaid on microdomain organization is IRBIT, setting a "threshold" for [IP(3)], assisting in fidelity of receptor specificity.     

Effects of rapid buffers on Ca2+ diffusion and Ca2+ oscillations.

Based on realistic mechanisms of Ca2+ buffering that include both stationary and mobile buffers, we derive and investigate models of Ca2+ diffusion in the presence of rapid buffers. We obtain a single transport equation for Ca2+ that contains the effects caused by both stationary and mobile buffers. For stationary buffers alone, we obtain an expression for the effective diffusion constant of Ca2+ that depends on local Ca2+ concentrations. Mobile buffers, such as fura-2, BAPTA, or small endogenous proteins, give rise to a transport equation that is no longer strictly diffusive. Calculations are presented to show that these effects can modify greatly the manner and rate at which Ca2+ diffuses in cells, and we compare these results with recent measurements by Allbritton et al. (1992). As a prelude to work on Ca2+ waves, we use a simplified version of our model of the activation and inhibition of the IP3 receptor Ca2+ channel in the ER membrane to illustrate the way in which Ca2+ buffering can affect both the amplitude and existence of Ca2+ oscillations.     

Novel function of phosphoinositide 3-kinase in T cell Ca2+ signaling. A phosphatidylinositol 3,4,5-trisphosphate-mediated Ca2+ entry mechanism

This study presents evidence that phosphoinositide (PI) 3-kinase is involved in T cell Ca(2+) signaling via a phosphatidylinositol 3,4, 5-trisphosphate PI(3,4,5)P(3)-sensitive Ca(2+) entry pathway. First, exogenous PI(3,4,5)P(3) at concentrations close to its physiological levels induces Ca(2+) influx in T cells, whereas PI(3,4)P(2), PI(4, 5)P(2), and PI(3)P have no effect on [Ca(2+)](i). This Ca(2+) entry mechanism is cell type-specific as B cells and a number of cell lines examined do not respond to PI(3,4,5)P(3) stimulation. Second, inhibition of PI 3-kinase by wortmannin and by overexpression of the dominant negative inhibitor Deltap85 suppresses anti-CD3-induced Ca(2+) response, which could be reversed by subsequent exposure to PI(3,4,5)P(3). Third, PI(3,4,5)P(3) is capable of stimulating Ca(2+) efflux from Ca(2+)-loaded plasma membrane vesicles prepared from Jurkat T cells, suggesting that PI(3,4,5)P(3) interacts with a Ca(2+) entry system directly or via a membrane-bound protein. Fourth, although D-myo-inositol 1,3,4,5-tetrakisphosphate (Ins(1,3,4, 5)P(4)) mimics PI(3,4,5)P(3) in many aspects of biochemical functions such as membrane binding and Ca(2+) transport, we raise evidence that Ins(1,3,4,5)P(4) does not play a role in anti-CD3- or PI(3,4,5)P(3)-mediated Ca(2+) entry. This PI(3,4,5)P(3)-stimulated Ca(2+) influx connotes physiological significance, considering the pivotal role of PI 3-kinase in the regulation of T cell function. Given that PI 3-kinase and phospholipase C-gamma form multifunctional complexes downstream of many receptor signaling pathways, we hypothesize that PI(3,4,5)P(3)-induced Ca(2+) entry acts concertedly with Ins(1,4,5)P(3)-induced Ca(2+) release in initiating T cell Ca(2+) signaling. By using a biotinylated analog of PI(3,4,5)P(3) as the affinity probe, we have detected several putative PI(3,4,5)P(3)-binding proteins in T cell plasma membranes.     

Interaction of caffeine-, IP3- and vanadate-sensitive Ca2+ pools in acinar cells of the exocrine pancreas

Summary Previous studies have shown the existence of functionally distinguishable inositol 1,4,5-trisphosphate- (IP₃) sensitive and IP₃-insensitive nonmitochondrial intracellular Ca²⁺ pools in acinar cells of the exocrine pancreas. For further characterization of Ca²⁺ pools, endoplasmic reticulum (ER) membrane vesicles were separated by Percoll gradient centrifugation which allowed us to distinguish five discrete fractions designatedP ₁ toP ₅ from the top to the bottom of the gradient. Measuring Ca²⁺ uptake and Ca²⁺ release with a Ca²⁺ electrode, we could differentiate three nonmitochondrial intracellular Ca²⁺ pools; (i) an IP₃-sensitive Ca²⁺ pool (IsCaP), vanadate- and caffeine-insensitive, (ii) a caffeine-sensitive Ca²⁺ pool (CasCaP), vanadate- and IP₃-insensitive, and (iii) a vanadate-sensitive Ca²⁺ pool (VasCaP), neither IP₃- nor caffeine-sensitive, into which Ca²⁺ uptake is mediated via a Ca²⁺ ATPase sensitive to vanadate at 10^–4 mol/liter. A fourth Ca²⁺ pool is neither IP₃- nor caffeine- or vanadate-sensitive. Percoll fractionP ₁ contained essentially the IsCaP, CasCaP and VasCaP and was mainly used for studies on Ca²⁺ uptake and Ca²⁺ release.When membrane vesicles were incubated in the presence of caffeine (2×10^–2 mol/liter), Ca²⁺ uptake up to the steady state [Ca²⁺] did not appear to be altered as compared to the control Ca²⁺ uptake. However, in control vesicles spontaneous Ca²⁺ release occurred after the steady state had been reached, whereas cfffeine-pretreated vesicles did not spontaneously release Ca²⁺. Addition of IP₃ at steady state [Ca²⁺] induced similar Ca²⁺ release followed by Ca²⁺ reuptake in both caffeine-pretreated and control vesicles. However, when caffeine was acutely added at steady state, Ca²⁺ was released from all Ca²⁺ pools including the IsCaP. Following Ca²⁺ reuptake after IP₃ had been added, a second addition of IP₃ to control vesicles induced further but smaller Ca²⁺ release, and a third addition resulted in a steady Ca²⁺ efflux by which all Ca²⁺ that had been taken up was released. This steady Ca²⁺ release started at a Ca²⁺ concentration between 5.5–8 ×10^–7 mol/liter and could also be induced by the IP₃ analogue inositol 1,4,5-trisphosphorothioate (IPS₃) or by addition of Ca²⁺ itself. Ruthenium red (10^–5 mol/liter) inhibited both caffeine-induced as well as Ca²⁺-induced but not IP₃-induced Ca²⁺ release. Heparin (100 g/m) inhibited IP₃-but not caffeine-induced Ca²⁺ release. The data indicate the presence of at least three separate Ca²⁺ pools in pancreatic acinar cells: the IsCaP, CasCaP and VasCaP. During Ca²⁺ uptake these Ca²⁺ pools appear to be separate. However, when steady state is reached, we assume that these Ca²⁺ pools come into contact and total Ca²⁺ release from all three pools can occur. The mechanism of this contact of Ca²⁺ pools is not clear but seems to be different from that induced by GTP in the presence of polyethylene glycol, which probably involves fusion of membranes.     

Effects of alkaline pH on sarcoplasmic reticulum Ca2+ release and Ca2+ uptake.

Alkalinization-induced Ca2+ release from isolated frog or rabbit sarcoplasmic reticulum vesicles appears to consist of two distinct components: 1) a direct activation of ruthenium red-sensitive Ca2+ release channels in terminal cisternae and 2) an increased ruthenium red-insensitive Ca2+ efflux through some other efflux pathway distributed throughout the sarcoplasmic reticulum. The first of these releases exhibits an alkalinization-induced inactivation process and does not depend on the ruthenium red-insensitive form of Ca2+ release as a triggering agent for secondary Ca(2+)-induced Ca2+ release. Both releases are inhibited when the extravesicular (i.e. cytoplasmic) free [Ca2+] is reduced. This may reflect an increased sensitivity of the Ca2+ release channels to Ca2+ at alkaline pH. The pH sensitivity of the ruthenium red-sensitive Ca2+ release channels could be of significance during excitation-contraction coupling. The ruthenium red-insensitive form of Ca2+ release is less likely to be physiologically relevant, but it probably has contributed greatly to reports of alkalinization-induced decreases in net sarcoplasmic reticulum Ca2+ uptake, particularly under conditions where oxalate supported Ca2+ uptake is much less affected, as here.     

Oxytocin regulates Ca2+ level in myometrium by influencing phosphoinositide metabolism

The effect of oxytocin on phosphoinositide metabolism as well as on membrane protein phosphorylation in myometrial tissue was studied. Oxytocin enhanced the 32P incorporation into phospholipids in myometrial tissue. The effect of oxytocin on phosphoinositide metabolism was also detected in plasma membrane of 20 days pregnant rats. Phosphorylated membrane lipids have been analysed and phosphatidylinositol 4, 5-bisphosphate proved to be the main reaction product. Oxytocin enhanced the 32P incorporation into phospholipids measured in the first 30 sec then the labeling decreased more rapidly then in case of the control. The effect of oxytocin proved to be concentration dependent. The protein phosphorylation was also influenced by oxytocin. However the amount of alkylphosphate formed depended on the presence or absence of Ca2+, Ca2+-calmodulin and cyclic AMP, oxytocin influenced the protein phosphorylation in the presence of Ca2+-calmodulin only.     

Gbetagamma dimers stimulate vascular L-type Ca2+ channels via phosphoinositide 3-kinase.

We have previously reported that, in venous myocytes, Gbetagamma scavengers inhibit angiotensin AT1A receptor-induced stimulation of L-type Ca2+ channels (1). Here, we demonstrate that intracellular infusion of purified Gbetagamma complexes stimulates the L-type Ca2+ channel current in a concentration-dependent manner. Additional intracellular dialysis of GDP-bound inactive Galphao or of a peptide corresponding to the Gbetagamma binding region of the beta-adrenergic receptor kinase completely inhibited the Gbetagamma-induced stimulation of Ca2+ channel currents. The gating properties of the channel were not affected by intracellular application of Gbetagamma, suggesting that Gbetagamma increased the whole-cell calcium conductance. In addition, both the angiotensin AT1A receptor- and the Gbetagamma-induced stimulation of L-type Ca2+ channels were blocked by pretreatment of the cells with wortmannin, at nanomolar concentrations. Correspondingly, intracellular infusion of an enzymatically active purified recombinant Gbetagamma-sensitive phosphoinositide 3-kinase, PI3Kgamma, mimicked Gbetagamma-induced stimulation of Ca2+ channels. Both Gbetagamma- and PI3Kgamma-induced stimulations of Ca2+ channel currents were reduced by protein kinase C inhibitors suggesting that the Gbetagamma/PI3Kgamma-activated transduction pathway involves a protein kinase C. These results indicate for the first time that Gbetagamma dimers stimulate the vascular L-type Ca2+ channels through a Gbetagamma-sensitive PI3K.     

Ca2+-induced polyphosphoinositide breakdown due to phosphomonoesterase activity in chicken erythrocytes.

Treatment of chicken erythrocytes with ionophore A23187 and Ca2+ caused the breakdown of a large proportion of the cellular polyphosphoinositides. Since no diacylglycerol or phosphatidate was generated, but there was a small increase in the level of phosphatidylinositol, it was concluded that breakdown occurred as a result of phosphomonoesterase activation. Experiments with subcellular fractions showed that the phosphomonoesterase activity was present in the cytosolic fraction of the cells.     

Effects of vasopressin and La3+ on plasma-membrane Ca2+ inflow and Ca2+ disposition in isolated hepatocytes. Evidence that vasopressin inhibits Ca2+ disposition.

Vasopressin caused a 40% inhibition of 45Ca uptake after the addition of 0.1 mM-45Ca2+ to Ca2+-deprived hepatocytes. At 1.3 mM-45Ca2+, vasopressin and ionophore A23187 each caused a 10% inhibition of 45Ca2+ uptake, whereas La3+ increased the rate of 45Ca2+ uptake by Ca2+-deprived cells. Under steady-state conditions at 1.3 mM extracellular Ca2+ (Ca2+o), vasopressin and La3+ each increased the rate of 45Ca2+ exchange. The concentrations of vasopressin that gave half-maximal stimulation of 45Ca2+ exchange and glycogen phosphorylase activity were similar. At 0.1 mM-Ca2+o, La3+ increased, but vasopressin did not alter, the rate of 45Ca2+ exchange. The results of experiments performed with EGTA or A23187 or by subcellular fractionation indicate that the Ca2+ taken up by hepatocytes in the presence of La3+ is located within the cell. The addition of 1.3 mM-Ca2+o to Ca2+-deprived cells caused increases of approx. 50% in the concentration of free Ca2+ in the cytoplasm [( Ca2+]i) and in glycogen phosphorylase activity. Much larger increases in these parameters were observed in the presence of vasopressin or ionophore A23187. In contrast with vasopressin, La3+ did not cause a detectable increase in glycogen phosphorylase activity or in [Ca2+]i. It is concluded that an increase in plasma membrane Ca2+ inflow does not by itself increase [Ca2+]i, and hence that the ability of vasopressin to maintain increased [Ca2+]i over a period of time is dependent on inhibition of the intracellular removal of Ca2+.     

Guanine nucleotide regulation of agonist binding to muscarinic cholinergic receptors. Relation to efficacy of agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization.

The efficacies of a series of six muscarinic cholinergic receptor agonists for stimulation of phosphoinositide breakdown and unidirectional efflux of 45Ca2+ in 1321N1 human astrocytoma cells were compared with the relative capacity of these agonists for formation of a GTP-sensitive high-affinity binding state in washed membranes. Carbachol and methacholine were 'full' agonists as regards phosphoinositide breakdown and Ca2+ mobilization, whereas bethanechol, arecoline and oxotremorine were 'partial' agonists for these two responses. Pilocarpine was the least efficacious of the six drugs tested. Except for pilocarpine, competition curves generated with the agonists and [3H]quinuclidinyl benzilate did not follow the Law of Mass Action for ligand interaction at a single site. Non-linear regression analyses of these data indicated that the data significantly better fit a two-, rather than a single-, site model with a high- and a low-affinity binding component. Competition curves generated in the presence of GTP were shifted to the right, and the extent of receptors in the high-affinity agonist-binding state was decreased. The relative efficacies of the six agonists for stimulation of phosphoinositide breakdown and Ca2+ mobilization were significantly correlated with the difference in affinities (KL/KH) between the two affinity states for each agonist. The relative efficacy of the agonists for stimulation of Ca2+ mobilization also was significantly correlated with the extent of receptors in the high-affinity state (%H) for each agonist. The results suggest that interaction with an as-yet unidentified guanine nucleotide regulatory protein is important in the mechanism whereby muscarinic receptors stimulate phosphoinositide breakdown in 1321N1 astrocytoma cells.     

Regulation of Ca2+ fluxes in rat liver mitochondria by Ca2+. Effects on Ca2+ distribution

Rat liver mitochondria are able to temporarily lower the steady-state concentration of external Ca²⁺ after having accumulated a pulse of added Ca²⁺. This has been attributed to inhibition of a putative -modulated efflux pathway [Bernardi, P. (1984)Biochim. Biophys. Acta 766, 277–282]. On the other hand, the rebounding could be due to stimulation of the uniporter by Ca²⁺ [Kröner, H. (1987)Biol. Chem. Hoppe-Seyler 369, 149–155]. By measuring unidirectional Ca²⁺ fluxes, it was found that the uniporter was stimulated during the rebounding peak both under Bernardi's and Kröner's conditions, while no effects on the efflux could be demonstrated. The rate of unidirectional efflux of Ca²⁺ was not affected by inhibition of the uniporter. It appears likely that the rebounding is due to stimulation of the uniporter rather than inhibition of efflux.     

Intracellular Ca2+ requirements for zymosan-stimulated phosphoinositide hydrolysis in mouse peritoneal macrophages

The phosphatidylinositol cycle has been demonstrated to be involved in the control of Ca2+ cytosolic levels in several cellular types. The Ca2+ requirements of phospholipase C activity and the described stimulation of phosphoinositide hydrolysis by Ca2+ ionophores make unclear the relationship between phosphatidylinositol cycle and Ca2+ mobilization. The results reported here suggest that intracellular Ca2+ is necessary for zymosan-stimulated phospholipase C activation in macrophages.     

Effect of GTP and Ca2+ on inositol 1,4,5-trisphosphate induced Ca2+ release from permeabilized rat exocrine pancreatic acinar cells

The effects of Ca2+ and GTP on the release of Ca2+ from the inositol 1,4,5-trisphosphate (IP3) sensitive Ca2+ compartment were investigated with digitonin permeabilized rat pancreatic acinar cells. The amount of Ca2+ released due to IP3 directly correlated with the amount of stored Ca2+ and was found to be inversely proportional to the medium free Ca2+ concentration. Ca2+ release induced by 0.18 microM IP3 was half maximally inhibited at 0.5 microM free Ca2+, i.e. at concentrations observed in the cytosol of pancreatic acinar cells. GTP did not cause Ca2+ release on its own, but a single addition of GTP (20 microM) abolished the apparent desensitization of the Ca2+ release which was observed during repeated IP3 applications. This effect of GTP was reversible. GTP gamma S could not replace GTP. Desensitization still occurred when GTP gamma S was added prior to GTP. The reported data indicate that GTP, stored Ca2+ and cytosolic free Ca2+ modulate the IP3 induced Ca2+ release.     

The role of phosphoinositide metabolism in Ca2+ signalling of skeletal muscle cells

• 1.1. The mobilization of Ca²⁺ from intracellular stores by d-myo-inositol 1,4,5-triphosphate[Ins(1,4,5)P₃] is now widely accepted as the primary link between plasma membrane receptors that stimulate phospholipase C and the subsequent increase in intracellular free Ca²⁺ that occurs when such receptors are activated (Berridge, 1993). Since the observations of VoIpe et al. (1985) which showed that Ins(1,4,5)P₃ could induce Ca²⁺ release from isolated terminal cisternae membranes and elicit contracture of chemically skinned muscle fibres, research has focused on the role of Ins(1,4,5)P₃ in the generation of SR Ca²⁺ transients and in the mechanism of excitation-contraction coupling (EC-coupling).
• 2.2. The mechanism of signal transduction at the triadic junction during EC-coupling is unknown. Asymmetric charge movement and mechanical coupling between highly specialized triadic proteins has been proposed as the primary mechanism for voltage-activated generation of SR Ca²⁺ signals and subsequent contraction. Ins(1,4,5)P₃ has also been proposed as the major signal transduction molecule for the generation of the primary Ca²⁺ transient produced during EC-coupling.
• 3.3. Investigations on the generation of Ca²⁺ transients by Ins(1,4,5)P₃ have been conducted on ion channels incorporated into lipid bilayers, skinned and intact fibres and isolated membrane vesicles. Ins(1,4,5)P₃ induces SR Ca²⁺ release and the enzymes responsible for its synthesis and degradation are present in muscle tissue. However, the sensitivity of the Ca²⁺ release mechanism to Ins(l,4,5)P₃ is highly dependent on experimental conditions and on membrane potential.
• 4.4. While Ins(1,4,5)P₃ may not be the major signal transduction molecule for the generation of the primary Ca²⁺ signal produced during voltage-activated contraction, this inositol polyphosphate may play a functional role as a modulator of EC-coupling and/or of the processes of myoplasmic Ca²⁺ regulation occurring on a time scale of seconds, during the events of contraction.

     

Effects of chronic administration of somatostatin on rat exocrine pancreas

We studied the effects of somatostatin on synthesis of pancreatic DNA, RNA and protein and on pancreatic weight and contents of DNA, protein, amylase and chymotrypsinogen in rats. In short term synthesis studies, rats were injected with 100 micrograms . kg-1 somatostatin or 0.15 M NaCl (control) at times 0, 8 and 16 h. Eight rats from each treatment group were killed 2, 4, 8, 12, 16, 20 and 24 h after beginning treatment. Incorporation rates in vivo of [3H]thymidine into DNA, [3H]uridine into RNA and [14C]phenylalanine into total protein were significantly depressed by somatostatin. In long term studies, four groups of 12 rats were injected every 8 h for 5 days with 0.15 M NaCl or 11, 33 or 100 micrograms . kg-1 somatostatin. Body weight was unaffected but pancreatic contents of DNA, protein and enzymes were significantly decreased by somatostatin. Administration of somatostatin inhibits DNA, RNA and protein synthesis in exocrine pancreas with resulting decreases in DNA and enzyme contents.     

Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration: studies on Ca2+ channels in rat parotid cells

1. Effects of Ca2+ agonist and antagonists on cytosolic free Ca2+ concentration [( Ca2+]i)were studied using quin2. 2. Nicardipine (NIC), diltiazem (DIL) and verapamil (VER) had no effect on the rise in [Ca2+]i evoked by carbachol. Methoxamine-elevated [Ca2+]i was inhibited by VER but not by NIC and DIL. 3. All Ca2+ antagonists tested produced a decline of [Ca2+]i elevated by isoproterenol to the resting level. 4. The addition of 30 mM K+ gradually elevated [Ca2+]i in normal and Ca2+-free media, but it did not increase 45Ca2+ uptake into cells. BAY K 8644 did not increase [Ca2+]i. 5. We suggest that voltage-sensitive Ca2+ channels are lacking and that at least 2 distinct receptor-operated Ca2+ channels exist in rat parotid cells.     

5-Hydroxytryptamine-induced phosphoinositide hydrolysis and Ca2+ mobilisation in canine cultured aorta smooth muscle cells.

The effect of 5-hydroxytryptamine (5-HT) on phospholipase C (PLC)-mediated phosphoinositide (PI) hydrolysis and intracellular Ca2+ ([Ca2+]i) changes was investigated in canine cultured aorta smooth muscle cells (ASMCs). 5-HT-stimulated inositol phosphate (IP) accumulation was time and concentration dependent with a half-maximal response (pEC50) and a maximal response at 6.4 and 10 microM, n = 6, respectively. Stimulation of ASMCs by 5-HT produced an initial transient peak followed by a sustained, concentration-dependent elevation in [Ca+]i. The half-maximal response (pEC50) values of 5-HT for the peak and sustained plateau were 7.1 and 6.9, respectively. Ketanserin and mianserin (1 and 3 nM), 5-HT2A antagonists, were equipotent and had high affinity in antagonising the 5-HT-induced IP accumulation and [Ca2+]i change with pK(B) values of 8.6-9.1 and 8.6-9.4, respectively. In contrast, the concentration-effect curves of 5-HT-induced IP and [Ca2+]i responses were not shifted until the concentrations of NAN-190 and metoctopramide (5-HT1A and 5-HT3 receptor antagonists, respectively) were increased to as high as 1 microM with pK(B) values of 5.7-6.3 and 6.1-6.6, respectively, indicating that the 5-HT receptor-mediated responses had low affinity for these antagonists. Pre-treatment of ASMCs with pertussis toxin (100 ng/mL, 24 h) caused a significant inhibition of 5-HT-induced IP accumulation and [Ca2+]i change in ASMCs. Depletion of external Ca2+ or removal of Ca2+ by addition of EGTA led to a significant attenuation of IP accumulation and [Ca2+]i change induced by 5-HT. Influx of external Ca2+ was required for the 5-HT-induced responses, because Ca2+-channel blockers--verapamil, nifedipine and Ni2+--partly inhibited the 5-HT-induced IP accumulation and Ca2+ mobilisation. The sustained elevation of [Ca2+]i response to 5-HT was dependent on the presence of external Ca2+. Removal of external Ca2+ by addition of 5 mM EGTA during the sustained phase caused a rapid decline in [Ca2+]i to lower than the resting level. The sustained elevation of [Ca2+]i could then be evoked by addition of 1.8 mM Ca2+ in the continued presence of 5-HT. These results demonstrate that 5-HT directly stimulates PLC-mediated PI hydrolysis and Ca2+ mobilisation, at least in part, through a pertussis toxin-sensitive G protein in canine ASMCs. 5-HT2A receptors may be predominantly mediating IP accumulation, and subsequently IP-induced Ca2+ mobilisation may function as the transducing mechanism for 5-HT-stimulated contraction of aorta smooth muscle.