Beta-adrenergic potentiation of endoplasmic reticulum Ca(2+) release in brown fat cells

We find that the adrenergic agonist isoproterenol increases intracellular Ca(2+) concentration ([Ca(2+)](i)) in cultured rat brown adipocytes. At the concentration used (10 microM), isoproterenol-induced Ca(2+) responses were sensitive to block by either alpha(1)- or beta-adrenergic antagonists, suggesting an interaction between these receptor subtypes. Despite reliance on beta-adrenoceptor activation, the Ca(2+) response was not due solely to increases in cAMP because, administered alone, the selective beta(3)-adrenergic agonist BRL-37344 or forskolin did not increase [Ca(2+)](i). However, increased cAMP elicited vigorous [Ca(2+)](i) increases in the presence of barely active concentrations of the alpha-adrenergic agonist phenylephrine or the P2Y receptor agonist UTP. Consistent with isoproterenol recruiting only inositol 1,4,5-trisphosphate (IP(3))-sensitive Ca(2+) stores, endoplasmic reticulum store depletion by thapsigargin blocked isoproterenol-induced Ca(2+) increases, but removal of external Ca(2+) did not. These results argue that increases in cAMP sensitize the IP(3)-mediated Ca(2+) release system in brown adipocytes.     

IRS-1 mediates inhibition of Ca2+ mobilization by insulin via the inhibitory G-protein Gi

Platelet agonists initiate aggregation and secretion by activating receptors coupled to the G-protein G(q), thereby raising cytosolic Ca(2+), [Ca(2+)](i). The rise in [Ca(2+)](i) is facilitated via inhibition of cAMP formation by the inhibitory G-protein of adenylyl cyclase, G(i). Since insulin attenuates platelet activation, we investigated whether insulin interferes with cAMP regulation. Here we report that insulin (0.5-200 nmol/liter) interferes with agonist-induced increases in [Ca(2+)](i) (ADP, thrombin), cAMP suppression (thrombin), and aggregation (ADP). The effects of insulin are as follows: (i) independent of the P2Y(12) receptor, which mediates ADP-induced cAMP lowering; (ii) not observed during G(s)-mediated cAMP formation; (iii) unaffected by treatments that affect phosphodiesterases (3-isobutyl-1-methylxanthine); and (iv) not changed by interfering with NO-mediated regulation of cAMP degradation (N(G)-monomethyl-l-arginine). Hence, insulin might interfere with G(i). Indeed, insulin induces the following: (i) tyrosine phosphorylation of the insulin receptor, the insulin receptor substrate-1 (IRS-1) and G(i)alpha(2); (ii) co-precipitation of IRS-1 with G(i)alpha(2) but not with other G alpha subunits. Despite persistent receptor activation, the association of IRS-1 with G(i)alpha(2) is transient, being optimal at 5 min and 1 nmol/liter insulin, which is sufficient to suppress Ca(2+) signaling by ADP, and at 10 min and 100 nmol/liter insulin, which is required to suppress Ca(2+) signaling by thrombin. Epinephrine, a known platelet sensitizer and antagonist of insulin, abolishes the effect of insulin on [Ca(2+)](i), tyrosine phosphorylation of G(i)alpha(2), and aggregation by interfering with the phosphorylation of the insulin receptor beta subunit. We conclude that insulin attenuates platelet functions by interfering with cAMP suppression through IRS-1 and G(i).     

Norepinephrine induces slow calcium signalling in murine brown preadipocytes through the beta-adrenoceptor/cAMP/protein kinase A pathway

The mechanism of adrenergically activated calcium signalling in isolated murine brown preadipocytes (stromal-vascular fraction) was studied with Fura-2. Norepinephrine (NE) generated in preadipocytes a slow Ca(2+)-response ( approximately 10 nM/min) without a burst and a maximum, whereas in mature brown adipocytes, the quick burst reached 1.5 microM [Ca(2+)](i). Thapsigargin, which is known to discharge Ca(2+) ions from the IP(3)-sensitive stores, initiated a huge capacitative calcium entry in mature brown adipocytes but failed to stimulate a response in preadipocytes. The beta-selective antagonist nadolol almost completely prevented the effect of NE on [Ca(2+)](i), while the antagonist of alpha-adrenoceptors phentolamine caused only a approximately 25% reduction of the cellular response. Forskolin or the cell-permeable Br-cAMP caused [Ca(2+)](i) rise, which were even higher than with NE. The protein kinase A (PKA) inhibitor N-[2-(p-bromocynnamylamino)ethyl]-5-isoquinolinesulfonamide (H-89) reduced and the phosphodiesterase inhibitors 3-isobutyl-1-methylxanthine (IBMX), N-cyclohexyl-N-(2-hydroxyethyl)-4-(6-(1,2-dihydro-2-oxoquinolyloxy))butyramide (OPC-3911), 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidone (Ro 20-1724) or the protein phosphatase inhibitor okadaic acid enhanced the NE-, isoproterenol- or forskolin-initiated cellular calcium responses. It was concluded that (i) brown preadipocytes lacked a trigger mechanism of initiation of [Ca(2+)](i) rises and (ii) the cAMP- and protein kinase A-mediated phosphorylation played an important role in the beta-adrenoceptor-initiated calcium signalling in these cells. All these features distinguish brown adipocyte precursors from differentiated brown adipocytes, where calcium signalling is initiated exclusively via alpha(1)-adrenoceptors and the trigger mechanism.     

Age-related changes in the control of hepatic cyclic AMP levels by alpha 1- and beta 2-adrenergic receptors in male rats

Hepatocytes from juvenile male rats (80-110 g) showed a 12-fold elevation of cAMP in response to epinephrine, which was mediated by beta 2-adrenergic receptors. In these cells, either alpha 1- or beta 2-adrenergic stimulation alone activated phosphorylase and glucose release although the alpha 1-phosphorylase response was 10-fold more sensitive to epinephrine and resulted in more rapid (by 10-20 s) activation of the enzyme. This suggests that the beta 2-adrenergic response is functionally unimportant for glycogenolysis, even in juvenile rats. beta 2-Adrenergic stimulation did, however, produce an increase in the rate of gluconeogenesis from [U-14C] lactate in these cells. Aging in the male rat was associated with attenuation of the beta 2-adrenergic cAMP response coupled with the emergence of an alpha 1-receptor-mediated accumulation of cAMP. The order of potency displayed by the alpha 1-adrenergic/cAMP system to adrenergic agonists and antagonists was identical with that of the alpha 1-adrenergic/Ca2+ system. These data suggest that, in maturity, hepatic alpha 1-receptors become linked to 2 separate transduction mechanisms, namely Ca2+ mobilization and cAMP generation. Calcium depletion of hepatocytes from adult, but not juvenile, male rats increased the alpha 1-component of the cAMP response to epinephrine, but under these conditions, alpha 1-activation of phosphorylase occurred more slowly than in calcium-replete cells. Blockade of alpha 2-adrenergic receptors did not significantly modify catecholamine effects on hepatocyte cAMP or phosphorylase a levels in male rats at any age studied, suggesting a lack of functional significance for these receptors in the regulation of glycogenolysis.     

β₃-Adrenergic activation of sequential Ca(2+) release from mitochondria and the endoplasmic reticulum and the subsequent Ca(2+) entry in rodent brown adipocytes

We studied how mitochondrial uncoupling by β(3)-adrenergic stimulation elicits Ca(2+) signals in rodent brown adipocytes by fluorometry of Ca(2+) concentrations ([Ca(2+)](i), [Ca(2+)](m) and [Ca(2+)](ER)) in the cytoplasm, mitochondria and the endoplasmic reticulum (ER), respectively, and mitochondrial membrane potential, using fura-2, rhod-5N, cameleon and rhodamine 123. Immunoblotting demonstrated α(1A)- and β(3)-adrenergic receptor and UCP1 in adipocytes, while RT-PCR revealed the mRNA of type 3, 7 and 9 adenylate cyclase, UCP1, UCP2, UCP3 and type 1 and 2 inositoltrisphosphate receptors. Isoproterenol and BRL37344, β-agonist, caused triphasic rises in [Ca(2+)](i) (β-responses) with mitochondrial depolarization in adipocytes. BRL37344 transiently decreased [Ca(2+)](m). β-Responses were blocked by propranolol, β-antagonist, H-89, protein kinase A blocker, and knockout of UCP1 gene. The late phase of β-responses was depressed by a Ca(2+) free, EGTA solution, U73122, a phospholipase C blocker, and thapsigargin, ER-Ca(2+) pump blocker, and by transfecting siRNA for type 2 IP(3)R. Intracellular loading of BAPTA/AM depressed the late phase more strongly than the initial phase. β-Agonists, phenylephrine, α-agonist, and cyclopiazonic acid, ER-Ca(2+) pump blocker, decreased [Ca(2+)](ER). Thus, the mitochondrial uncoupling by β(3)-adrenergic activation causes Ca(2+) release from mitochondria and subsequently from the ER and further evokes plasmalemmal Ca(2+) entries, including the store-operated Ca(2+) entry.     

Analysis of inhibition by H89 of UCP1 gene expression and thermogenesis indicates protein kinase A mediation of beta(3)-adrenergic signalling rather than beta(3)-adrenoceptor antagonism by H89

Although it has generally been assumed that protein kinase A (PKA) is essential for brown adipose tissue function, this has not as yet been clearly demonstrated. H89, an inhibitor of PKA, was used here to inhibit PKA activity. In cell extracts, it was confirmed that norepinephrine stimulated PKA activity, which was abolished by H89 treatment. In isolated brown adipocytes, H89 inhibited adrenergically induced thermogenesis (with an IC(50) of approx. 40 microM), and in cultured cells, adrenergically stimulated expression of the uncoupling protein-1 (UCP1) gene was abolished by H89 (full inhibition with 50 microM). However, H89 has been reported to be an adrenergic antagonist on beta(1)/beta(2)-adrenoceptors (AR). Although adrenergic stimulation of thermogenesis and UCP1 gene expression are mediated via beta(3)-ARs, it was deemed necessary to investigate whether H89 also had antagonistic potency on beta(3)-ARs. It was found that EC(50) values for beta(3)-AR-selective stimulation of cAMP production (with BRL-37344) in brown adipose tissue membrane fractions and in intact cells were not affected by H89. Similarly, the EC(50) of adrenergically stimulated oxygen consumption was not affected by H89. As H89 also abolished forskolin-induced UCP1 gene expression, and potentiated selective beta(3)-AR-induced cAMP production, H89 must be active downstream of cAMP. Thus, no antagonism of H89 on beta(3)-ARs could be detected. We conclude that H89 can be used as a pharmacological tool for elucidation of the involvement of PKA in cellular signalling processes regulated via beta(3)-ARs, and that the results are concordant with adrenergic stimulation of thermogenesis and UCP1 gene expression in brown adipocytes being mediated via a PKA-dependent pathway.     

Adrenergic regulation of adipocyte metabolism

Adipocytes can be readily isolated from intact adipose tissue. In adipocytes from hamster and human white adipose tissue it is possible to demonstrate beta, alpha 1, and alpha 2 adrenoceptors. Alpha 2 adrenoceptor activation inhibits while beta adrenoceptor activation stimulates cyclic AMP accumulation and lipolysis. The effects of catecholamines on cyclic AMP accumulation are mediated through regulation of adenylate cyclase activity, which is activated through beta adrenoceptors and inhibited through alpha 2 adrenoceptors. Activation of alpha 1 adrenergic receptors has been shown to be associated with elevations of cytosol calcium and increased turnover of phosphatidylinositol. In white adipocytes, the only known alpha 1 adrenergic effects are inhibition of glycogen synthase and stimulation of glycogen phosphorylase via mechanisms distinct from those by which cyclic AMP produces similar end effects. In brown adipocytes, alpha 1 adrenoceptor activation stimulates respiration. Thyroid hormones primarily regulate the sensitivity of adipocytes to beta-adrenergic amines while having little effect on alpha adrenoceptor sensitivity.     

A role for protein kinase C during rat egg activation

Upon sperm-egg interaction, an increase in intracellular calcium concentration ([Ca(2+)](i)) is observed. Several studies reported that cortical reaction (CR) can be triggered not only by a [Ca(2+)](i) rise but also by protein kinase C (PKC) activation. Because the CR is regarded as a Ca(2+)-dependent exocytotic process and because the calcium-dependent conventional PKCs (cPKC) alpha and beta II are considered as exocytosis mediators in various cell systems, we chose to study activation of the cPKC in the rat egg during in vivo fertilization and parthenogenetic activation. By using immunohistochemistry and confocal microscopy techniques, we demonstrated, for the first time, the activation of the cPKC alpha, beta I, and beta II during in vivo fertilization. All three isozymes examined presented translocation to the egg's plasma membrane as early as the sperm-binding stage. However, the kinetics of their translocation was not identical. Activation of cPKC alpha was obtained by the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) or by 1-oleoyl-2-acetylglycerol (OAG) but not by the calcium ionophore ionomycin. PKC alpha translocation was first detected 5-10 min after exposure to TPA and reached a maximum at 20 min, whereas in eggs activated by OAG, translocation of PKC alpha was observed almost immediately and reached a maximum within 5 min. These results suggest that, although [Ca(2+)](i) elevation on its own does not activate PKC alpha, it may accelerate OAG-induced PKC alpha activation. We also demonstrate a successful inhibition of the CR by a myristoylated PKC pseudosubstrate (myrPKCPsi), a specific PKC inhibitor. Our study suggests that exocytosis can be triggered independently either by a [Ca(2+)](i) rise or by PKC.     

alpha(2A)-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels

alpha(2A)-Adrenergic receptor-mediated Ca(2+) signaling and integrin alpha(IIb)beta(3) exposure were investigated in human platelets under conditions where indirect, thromboxane- or ADP-mediated effects were absent. The alpha(2)-adrenergic receptor agonists, UK14304 and epinephrine (EPI), were unable to raise cytosolic levels of inositol 1,4,5-trisphosphate (InsP(3)) or Ca(2+) but potentiated the [Ca(2+)](i) rises evoked by other agonists that act through stimulation of phospholipase C (thrombin or platelet-activating factor) or stimulation of Ca(2+)-induced Ca(2+) release (CICR) in the absence of InsP(3) generation (thimerosal or thapsigargin). In addition, alpha(2)-adrenergic stimulation resulted in a 20% lowering in the cytosolic cAMP level. In platelets treated with G(salpha)-stimulating prostaglandin E(1), EPI increased the Ca(2+) signal evoked by either phospholipase C- or CICR-stimulating agonists mainly through modulation of the cAMP level. The stimulating effects of UK14304 and EPI on platelet Ca(2+) responses, and also on integrin alpha(IIb)beta(3) exposure and platelet aggregation, were abolished by pharmacological stimulation of cAMP-dependent protein kinase, and these effects were mimicked by inhibition of this activity. In permeabilized platelets, UK14304 and EPI potentiated InsP(3)-induced, CICR-mediated mobilization of Ca(2+) from internal stores in a similar way as did inhibition of cAMP-dependent protein kinase. In summary, a G(ialpha)-mediated decrease in cAMP level appears to play a major role in the platelet-activating effects of alpha(2A)-adrenergic receptor stimulation. Thus, in platelets, unlike other cell types, occupation of the G(ialpha)-coupled alpha(2A)-adrenergic receptors does not result in phospholipase C activation but rather in modulation of the Ca(2+) response by relieving cAMP-mediated suppression of InsP(3)-dependent CICR.     

Albumin-bound lipids induce free cytoplasmic calcium oscillations in human osteoblast-like cells

[Ca(2+)](i) oscillations were found in human osteoblast-like cells (hOB cells) exposed to high-lipid bovine serum albumin (BSA), but not when exposed to low-lipid BSA. These [Ca(2+)](i) oscillations were inhibited by heptanol and suramin, which implies that gap junctions and purinergic signalling may be important for these [Ca(2+)](i) oscillations. The high-lipid BSA preparation that was used contains arachidonic acid. [Ca(2+)](i) oscillations could be induced by low lipid albumin with arachidonic acid added. The albumin-bound lipids were also important for osteoblast growth since DNA synthesis and the total cell protein content was higher in hOB cells exposed to high-lipid BSA. The effect of arachidonic acid on hOB cell proliferation was bone-donor dependent; both stimulatory and inhibitory effects were observed. The physiological importance of albumin-bound lipids is unclear; given that albumin has only minimal contact with osteoblasts under normal conditions. Only when bone capillaries are disrupted, e.g. during a fracture, would significant amounts of albumin reach osteoblasts. Albumin-bound lipids could therefore contribute to stimulation of osteoblast proliferation during fracture healing.     

Phosphatase inhibitor cantharidin blocks adenosine A(1) receptor anti-adrenergic effect in rat cardiac myocytes

Experiments were performed to examine whether the protein phosphatase inhibitor cantharidin blocks the anti-adrenergic effect of adenosine A(1) receptor stimulation. In electrically stimulated adult rat ventricular myocytes loaded with the intracellular calcium concentration ([Ca(2+)](i)) indicator fluo-3, isoproterenol (10 nM) increased systolic [Ca(2+)](i) by 46%, increased twitch amplitude by 56%, and increased total cellular cAMP content by 140%. The adenosine A(1) receptor agonist 2-chloro-N(6)-cyclopentlyadenosine (CCPA) reduced isoproterenol-stimulated [Ca(2+)](i) and contractility by 87 and 80%, respectively, but reduced cAMP content by only 18%. Cantharidin had no effects on myocyte [Ca(2+)](i), contractility, or cAMP in the absence or presence of isoproterenol but blocked the effects of CCPA on [Ca(2+)](i) and contractility by approximately 44%. Cantharidin had no effect on CCPA attenuation of isoproterenol-induced increases in cAMP. Pretreatment with CCPA also reduced the increase in contractile parameters produced by the direct cAMP-dependent protein kinase A (PKA) activator 8-bromocAMP. These results suggest that activation of protein phosphatases mediate, in part, the anti-adrenergic effect of adenosine A(1) receptor activation in ventricular myocardium.     

Inhibition of ADP-induced intracellular Ca2+ responses and platelet aggregation by the P2Y12 receptor antagonists AR-C69931MX and clopidogrel is enhanced by prostaglandin E1

P2Y(12) antagonists such as clopidogrel and AR-C69931MX inhibit aggregation by antagonizing the effects of ADP at P2Y(12) receptors on platelets. Agents such as PGE(1) also inhibit aggregation by stimulating adenylate cyclase to produce cAMP, which interferes with Ca(2+) mobilization within the cell. Since one facet of P2Y(12) receptors is that they mediate inhibition of adenylate cyclase by ADP, it might be expected that P2Y(12) antagonists would interact with PGE(1). We have explored the effects of PGE(1) and AR-C69931MX singly and in combination on ADP-induced intracellular Ca(2+) ([Ca(2+)](i)) responses and aggregation. PGE(1) alone caused parallel dose-dependent inhibition of [Ca(2+)](i) and aggregation responses. AR-C66931MX alone caused only partial inhibition of [Ca(2+)](i) despite a marked inhibitory effect on aggregation. Combinations of PGE(1) with AR-C66931MX were found to act in synergy to reduce both [Ca(2+)](i) and aggregation. This effect was confirmed in patients with acute coronary syndromes by studying the inhibitory effects of PGE(1) on [Ca(2+)](i) and aggregation before and after clopidogrel. In summary, we have shown that P2Y(12) antagonists interact with natural agents such as PGE(1) to provide more effective inhibition of [Ca(2+)](i) and platelet aggregation. This would contribute to the effectiveness of P2Y(12) antagonists as antithrombotic agents in man.     

Involvement of alpha7 nicotinic acetylcholine receptors in activation of tyrosine hydroxylase and dopamine beta-hydroxylase gene expression in PC12 cells

Nicotine treatment increases intracellular free Ca(2+) concentration [Ca(2+)](i), stimulates catecholamine release, and elevates gene expression for the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH). However, the type of nicotinic acetylcholine receptors (nAChRs) mediating these events is unclear. The nAChR receptor antagonists alpha-bungarotoxin (alphaBTX) and methyllycaconitine greatly reduced the nicotine-triggered initial transient rise in [Ca(2+)](i) and prevented the second prolonged elevation of [Ca(2+)](i), suggesting the involvement of alpha7 nAChRs. Two specific alpha7 nicotinic agonists, 3-(2,4-dimethoxybenzilidene)anabaseine (DMXB) and E, E-3-(cinnamylidene)anabaseine (3-CA), were found to elicit a small, delayed increase in [Ca(2+)](i) with kinetics and magnitude similar to the second elevation observed with nicotine. This increase was inhibited by the inositol trisphosphate receptor antagonist xestospongin C. Exposure to 3-CA or DMXB for 6 or 24 h elevated TH and DBH mRNA levels two- to fourfold over control levels. These agonists were more effective than nicotine alone in increasing TH and DBH gene expression and significantly elevated [Ca(2+)](i) for up to 6 h. The increase in [Ca(2+)](i) or the elevation in TH mRNA by 3-CA was completely inhibited by alphaBTX. This study, for the first time, implicates stimulation of alpha7 nAChRs in the activation of TH and DBH gene expression.     

Mechanisms of leptin secretion from white adipocytes

The mechanisms regulating leptin secretion were investigated in isolated rat white adipocytes. Insulin (1-100 nM) linearly stimulated leptin secretion from incubated adipocytes for at least 2 h. The adrenergic agonists norepinephrine, isoproterenol (two nonselective beta-agonists), or CL-316243 (potent beta3) all inhibited insulin (10 nM)-stimulated leptin release. The inhibitory effects of norepinephrine and isoproterenol could be reversed not only by the nonselective antagonist propranolol but also by the selective antagonists ICI-89406 (beta1) or ICI-118551 (beta2), the beta2-antagonist being less effective than the beta1. Insulin-stimulated leptin secretion could also be inhibited by a series of agents increasing intracellular cAMP levels, such as lipolytic hormones (ACTH and thyrotropin-stimulating hormone), various nonhydrolyzable cAMP analogs, pertussis toxin, forskolin, methylxanthines (caffeine, theophylline, IBMX), and specific inhibitors of phosphodiesterase III (imazodan, milrinone, and amrinone). Significantly, antilipolytic agents other than insulin (adenosine, nicotinic acid, acipimox, and orthovanadate) did not mimic the acute stimulatory effects of insulin on leptin secretion under these conditions. We conclude that norepinephrine specifically inhibits insulin-stimulated leptin secretion not only via the low-affinity beta3-adrenoceptors but also via the high-affinity beta1/beta2-adrenoceptors. Moreover, it is suggested that 1) activation of phosphodiesterase III by insulin represents an important metabolic step in stimulation of leptin secretion, and 2) lipolytic hormones competitively counterregulate the stimulatory effects of insulin by activating the adenylate cyclase system.     

Somatostatin: a metabolic regulator

Somatostatin, the hypothalamic release-inhibiting factor, has been found to stimulate gluconeogenesis in rat kidney cortical slices. Stimulation by somatostatin was linear and dose-dependent. Other bioactive peptides such as cholecystokinin, gastrointestinal peptide, secretin, neurotensin, vasoactive intestinal peptide, pancreatic polypeptide, beta endorphin and substance P did not affect the renal gluconeogenic activity. Somatostatin-induced gluconeogenesis was blocked by phentolamine (alpha adrenergic antagonist) and prazosin (alpha1 adrenergic antagonist) but not by propranolol (beta adrenergic antagonist) and yohimbine (alpha2 adrenergic antagonist) suggesting that the effect is via alpha1 adrenergic stimuli. Studies on the involvement of Ca2+ revealed that tissue depletion and omission of Ca2+ from the reaction mixture would abolish the stimulatory effect of somatostatin. Furthermore, somatostatin enhanced the uptake of 45calcium in renal cortical slices which could be blocked by lanthanum, an inhibitor of Ca2+ influx. It is proposed that the stimulatory effect of somatostatin on renal gluconeogenesis is mediated by alpha1 adrenergic receptors, or those which functionally resemble alpha1 receptors and that the increased influx of Ca2+ may be the causative factor for carrying out the stimulus.     

Modulation of intracellular Ca(2+) via alpha(1B)-adrenoreceptor signaling molecules,G alpha(h) (transglutaminase II) and phospholipase C-delta 1

We characterized the alpha(1B)-adrenoreceptor (alpha(1B)-AR)-mediated intracellular Ca(2+) signaling involving G alpha(h) (transglutaminase II, TGII) and phospholipase C (PLC)-delta 1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to activation of the alpha(1B)-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-delta 1 substantially reduced both the peak and sustained levels of [Ca(2+)](i). Expression of TGII mutants lacking the interaction with PLC-delta 1 resulted in a reduced capacitative Ca(2+) entry. Reduced expression of PLC-delta 1 displayed a transient elevation of [Ca(2+)](i) and a reduction in capacitative Ca(2+) entry. Expression of the C2-domain of PLC-delta 1, which contains the TGII interaction site, resulted in reduction of the alpha(1B)-AR-evoked peak increase in [Ca(2+)](i), while the sustained elevation in [Ca(2+)](i) and capacitative Ca(2+) entry remained unchanged. These findings demonstrate that stimulation of PLC-delta 1 via coupling of the alpha(1B)-AR with TGII evokes both Ca(2+) release and capacitative Ca(2+) entry and that capacitative Ca(2+) entry is mediated by the interaction of TGII with PLC-delta 1.     

Essential role of calcium influx in the adrenergic regulation of cAMP and cGMP in rat pinealocytes

The role of Ca2+ in the adrenergic stimulation of pinealocyte cAMP and cGMP was investigated. In this tissue alpha 1-adrenoceptor activation, which by itself is without effect, potentiates beta 1-adrenergic stimulation of cAMP and cGMP 30- to 100-fold. The present results indicate that chelation of extracellular Ca2+ with EGTA or inhibition of Ca2+ influx with inorganic Ca2+ channel blockers (La3+, Co2+, Mn2+) markedly reduces the cyclic nucleotide response to norepinephrine, a mixed alpha 1- and beta-adrenergic agonist, but not to isoproterenol, a beta-adrenergic agonist. In addition, the potentiating effects of alpha 1-adrenergic agonists were mimicked by agents which elevate cytosolic Ca2+, including K+ (EC50 = 2 X 10(-2) M), ouabain (EC50 = 2 X 10(-6) M), ionomycin (EC50 = 3 X 10(-6) M), and A23187 (EC50 = 2 X 10(-6) M); each potentiated the effects of beta-adrenergic stimulation but had no effect alone. Together these results indicate that an alpha 1-adrenoceptor-stimulated Ca2+ influx is essential for norepinephrine to increase pinealocyte cAMP and cGMP.