Activation of alpha 2-adrenergic receptors decreases Ca2+ influx to inhibit insulin secretion in a hamster beta-cell line: an action mediated by a guanosine triphosphate-binding protein.

Activation of the sympathetic nervous system inhibits insulin secretion. We tested the hypothesis that activation of alpha 2-adrenergic receptors on the beta-cell by epinephrine or clonidine attenuates insulin release by an effect on the voltage-dependent Ca2+ channel (VDCC) and examined the role of G-proteins in this signal transduction pathway. Using a cultured SV40-transformed hamster beta-cell line (HIT cells) as a model system, we determined the effect of alpha 2-adrenergic agonists on insulin secretion, 86Rb+ efflux (a marker for K+ channel flux), and the free cytosolic Ca2+ level [( Ca2+]i) monitored in fura-2-loaded cells. In a dose-dependent manner, epinephrine and clonidine (10(-8)-10(-5)M) attenuated the increase in [Ca2+]i and insulin secretion induced by either K+ depolarization or stimulation of the VDCC with the agonist Bay K 8644. Epinephrine failed to affect the rise in [Ca2+]i induced by carbamylcholine, an agent that mobilizes intracellular Ca2+. Epinephrine also did not changes 86Rb+ efflux from HIT cells. The inhibitory effects of epinephrine were prevented by the alpha 2-adrenergic antagonist idazoxan, but were unaffected by the alpha 1-adrenergic antagonist phenoxybenzamine. Pretreatment of HIT cells with pertussis toxin (0.1 micrograms/ml) overnight abolished the inhibitory effects of epinephrine and clonidine on both [Ca2+]i and insulin secretion. These data suggest that one mechanism by which alpha 2-adrenergic agonists inhibit insulin secretion is by inhibiting Ca2+ influx through VDCC, an action that is mediated through a pertussis toxin-sensitive G-protein.     

Expression of fibrinogen receptors during activation and subsequent desensitization of human platelets by epinephrine

Epinephrine causes platelet aggregation and secretion by interacting with alpha 2-adrenergic receptors on the platelet surface. Platelet aggregation requires the binding of fibrinogen to a specific receptor on the membrane glycoprotein IIb-IIIa complex. Although the IIb-IIIa complex is identifiable on the surface of resting platelets, the fibrinogen receptor is expressed only after platelet activation. The current studies were designed to examine the effect of occupancy of platelet alpha 2-adrenergic receptors by epinephrine on the expression of fibrinogen receptors and on the aggregation of platelets. The ability of epinephrine to induce the expression of fibrinogen receptors was studied under two different conditions: acute stimulation (less than 1 min) and prolonged stimulation (50 to 90 min), the latter of which is associated with a reduction or "desensitization" of the platelet aggregation response. Expression of the fibrinogen receptor was monitored with 125I-fibrinogen as well as with 125I-PAC-1 (PAC-1), a monoclonal antibody that binds to the glycoprotein IIb-IIIa complex only after platelets are activated. Epinephrine caused an immediate increase in PAC-1 and fibrinogen binding that was dependent on occupancy of the alpha 2-receptor by epinephrine and on the presence of extracellular free Ca (KCa = 30 mumol/L). By itself, 1 mmol/L Mg was unable to support induction of the fibrinogen receptor by epinephrine. However, it did decrease the Ca requirement by about two orders of magnitude. Prolonged stimulation of unstirred platelets by epinephrine led to a 70% decrease in the aggregation response when the platelets were subsequently stirred. Despite their decreased aggregation response, desensitized platelets bound PAC-1 and fibrinogen normally, indicating that the loss of aggregation was not due simply to a decrease in fibrinogen receptor expression. Although desensitization was not affected by pretreatment of the platelets with aspirin, it was partially prevented when extracellular Ca was chelated by EDTA during the long incubation with epinephrine. These studies demonstrate that once platelet alpha 2-adrenergic receptors are occupied by epinephrine, extracellular Ca is involved in initiating the aggregation response by supporting the induction of the fibrinogen receptor and the binding of fibrinogen. Furthermore. Ca-dependent reactions subsequent to fibrinogen binding may be necessary for maximal platelet aggregation and are impaired when platelets become desensitized to epinephrine.     

Differential requirements for platelet aggregation and inhibition of adenylate cyclase by epinephrine. Studies of a familial platelet alpha 2-adrenergic receptor defect

We describe a family whose members have impaired platelet aggregation and secretion responses to epinephrine with normal responses to adenosine diphosphate and collagen. Platelet alpha 2-adrenergic receptors (measured using 3H methyl-yohimbine) were diminished in the propositus (78 sites per platelet), his two sisters (70 and 27 sites per platelet), and parents (37 and 63 sites per platelet), but not in two maternal aunts (12 normal subjects, 214 +/- 18 sites per platelet; mean +/- SE). However, the inhibition of cyclic adenosine monophosphate (cAMP) levels by epinephrine in platelets exposed to 400 nmol/L PGI2 was similar in the patients and five normal subjects (epinephrine concentration for 50% inhibition, 0.04 +/- 0.01 mumol/L v 0.03 +/- 0.01 mumol/L; P greater than .05). In normal platelets, the concentration of yohimbine (0.18 mumol/L) required for half maximal inhibition of aggregation induced by 2 mumol/L epinephrine was lower than that for inhibition of its effect on adenylate cyclase (1.6 mumol/L). In quin2 loaded platelets, thrombin (0.1 U/mL) stimulated rise in cytoplasmic Ca2+ concentration, [Ca2+]i, was normal in the two patients studied. The PGI2 analog ZK 36,374 completely inhibited thrombin-induced rise in [Ca2+]i; the reversal of this inhibition by epinephrine was normal in the two patients. Thus, despite the impaired aggregation response to epinephrine, platelets from these patients have normal ability to inhibit PGI2-stimulated cAMP levels. These patients with an inherited receptor defect provide evidence that fewer platelet alpha 2-adrenergic receptors are required for epinephrine-induced inhibition of adenylate cyclase than for aggregation.     

Deficient elevation of the cytoplasmic calcium ion concentration by epinephrine in epinephrine-insensitive platelets of patients with myeloproliferative disorders

Elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) by epinephrine and epinephrine-induced inhibition of prostaglandin E1 (PGE1)-stimulated cyclic adenosine monophosphate (cAMP) accumulation were assessed in platelets from three groups of subjects; normal controls (NS, n = 11) and patients with myeloproliferative disorders whose platelets were either sensitive (ES, n = 9) or specifically insensitive (El, n = 7) to the aggregatory effect of epinephrine. The inhibition by epinephrine of cAMP accumulation in the platelets exposed to 500 nM PGE1 was not significantly different between the three groups. Therefore, despite the defective aggregation response to epinephrine, platelets from the El group seemed to retain normal response, which was attained through alpha 2-adrenergic receptors, guanine nucleotide binding regulatory protein, and the adenylate cyclase system. However, in aequorin-loaded, washed platelets, the epinephrine-stimulated rise in [Ca2+]i showed significant decrease in the El group compared with the other groups (P less than 0.01). Thus the mechanism for the impaired aggregation response to epinephrine in platelets from the El group could include the defect that exists in the pathway from receptor binding of epinephrine to the aggregation response through [Ca2+]i elevation.     

Acute vigorous exercise primes enhanced NO release in human platelets

Activation of platelets by acute vigorous exercise has been demonstrated by various parameters, including an increase in agonist-induced platelet [Ca2+]i levels. However, direct evidence is lacking regarding how acute exercise affects platelet-derived NO. Twenty-three healthy male non-smokers (21-59 years) underwent a symptom-limited treadmill exercise test. Washed platelets were prepared from blood samples obtained before and immediately after exercise. All subjects completed at least Bruce stage 2 and were each negative for ischemia. With a low dose (2 microg/ml) of collagen, NO release from washed platelets, detected by the NO-selective microelectrode, was significantly increased after exercise (pmols/10(8) platelets, before: 0.64+/-0.11, after: 1.03+/-0.18; P<0.005) without changes in aggregation ability. This enhanced NO release was accompanied by increased platelet [Ca2+]i levels (before: 232+/-25, after: 296+/-37; P<0.01). With a high dose (5 or 10 microg/ml) of collagen, NO release and aggregation were both modestly, but significantly, enhanced after exercise. The exercise-induced enhancement of platelet NO release in response to collagen was also suggested by increase in platelet cyclic guanosine monophosphate accumulation and augmenting effect of N(G)-monomethyl-L-arginine on platelet aggregation. In summary, acute strenuous exercise primes enhanced NO release and may play a protective role against exercise-induced activation of platelets in normal subjects.     

Abnormalities of cytoplasmic Ca2+ in platelets from patients with uremia

Uremic patients have a hemorrhagic tendency, often associated with prolonged bleeding times and decreased platelet function in vitro. Whether these defects result from abnormalities in plasma factors affecting platelet activity, platelet surface receptors, intracellular platelet mediators, or other aspects of platelet behavior is unknown. To examine the possibility that the abnormality in platelet function may result from aberrations in Ca2+ homeostasis, blood was obtained from 29 patients with severe uremia. The platelets were washed, loaded with the Ca2+ -sensitive probes indo-1 and aequorin, gel-filtered, and resuspended in either plasma or buffer. Of the 29 patients, seven had template bleeding times prolonged to 11 minutes or more, but platelet aggregation in plasma was not consistently impaired in these patients. However, in aequorin-loaded platelets from the patients with long bleeding times, the highest elevation of cytoplasmic calcium [( Ca2+]i) in response to the Ca2+ ionophore A23187, arachidonate, adenosine diphosphate (ADP), or epinephrine was lower than that seen in platelets from both uremic patients with less prolonged bleeding times and normal volunteers. The reduced [Ca2+]i response was associated with decreased aggregation of gel-filtered platelets suspended in buffer. Suspending washed aequorin-loaded uremic platelets in normal plasma for 20 minutes did not reverse the decreased agonist-induced rise in [Ca2+]i; platelets from a normal donor resuspended in uremic plasma aggregated and produced a normal increase in [Ca2+]i in response to agonists. We conclude that the platelet defect seen in some patients with uremia is associated with a decreased rise in platelet [Ca2+]i after stimulation and that this is a manifestation of an intrinsic platelet defect.     

The periodontal pathogen Porphyromonas gingivalis sensitises human blood platelets to epinephrine

Recent studies indicate connections between periodontitis and atherothrombosis, and the periodontal pathogen Porphyromonas gingivalis has been found within atherosclerotic lesions. P. gingivalis-derived proteases, designated gingipains activate human platelets, probably through a "thrombin-like" activity on protease-activated receptors (PARs). However, the potential interplay between P. gingivalis and other physiological platelet activators has not been investigated. The aim of this study was to elucidate consequences and mechanisms in the interaction between P. gingivalis and the stress hormone epinephrine. By measuring changes in light transmission through platelet suspensions, we found that P. gingivalis provoked aggregation, whereas epinephrine alone never had any effect. Intriguingly, pre-treatment of platelets with a low, sub-threshold number of P. gingivalis (i.e. a density that did not directly provoke platelet aggregation) resulted in a marked aggregation response when epinephrine was added. This synergistic action was not inhibited by the cyclooxygenas inhibitor aspirin. Furthermore, fura-2-measurements revealed that epinephrine caused an intracellular Ca(2+) mobilization in P. gingivalis pre-treated platelets, whereas epinephrine alone had no effect. Inhibition of the arg-specific gingipains, but not the lys-specific gingipains, abolished the aggregation and the Ca(2+) response provoked by epinephrine. Similar results were achieved by separate blockage of platelet alpha(2)-adrenergic receptors and PARs. In conclusion, the present study shows that a sub-threshold number of P. gingivalis sensitizes platelets to epinephrine. We suggest that P. gingivalis-derived arg-specific gingipains activates a small number of PARs on the surface of the platelets. This leads to an unexpected Ca(2+) mobilization and a marked aggregation response when epinephrine subsequently binds to the alpha(2)-adrenergic receptor. The present results are consistent with a direct connection between periodontitis and stress, and describe a novel mechanism that may contribute to pathological platelet activation.     

Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine.

Human platelets stimulated by epinephrine undergo enhanced turnover of phosphatidylinositol 4,5-bisphosphate, accumulate inositol trisphosphate, diacylglycerol, and phosphatidic acid, and phosphorylate a 47-kDa protein. All of these phenomena indicate stimulation of phospholipase C. These responses are blocked completely by inhibitors of alpha 2-adrenergic receptors (yohimbine), cyclooxygenase (aspirin or indomethacin), phospholipase A [2-(p-amylcinnamoyl)amino-4-chlorobenzoic acid (ONO-RS-082)], Na+/H+ exchange [ethylisopropylamiloride (EIPA)], fibrinogen binding to glycoprotein IIb/IIIa (antibody A2A9), Ca2+/Mg+ binding (EDTA), or removal of fibrinogen. Epinephrine evokes (i) an increased turnover of ester-linked arachidonic acid in aspirin treated platelets that is inhibited by ONO-RS-082, EDTA, yohimbine, or the absence of fibrinogen and (ii) a rapid cytoplasmic alkalinization that is inhibited partially by blockage of cyclooxygenase activity and completely by A2A9 or EIPA. In contrast, when incubated with subaggregatory concentrations of the prostaglandin H2/thromboxane A2 analogue [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic acid (U46619) and epinephrine, aspirin-treated platelets show a potentiation of phospholipase C activation that is unaffected by the above inhibitors. We propose that epinephrine, in promoting exposure of glycoprotein IIb/IIIa sites for fibrinogen binding, leads to a cytoplasmic alkalinization, which, in conjunction with local shifts in Ca2+, promotes low-level activation of phospholipase A. The resulting free arachidonic acid is converted to cyclooxygenase products, which, potentiated by epinephrine, activate phospholipase C. This further amplifies the initial stimulatory response.     

Cytosolic free calcium in platelets: relationships to blood pressure and indices of systemic calcium metabolism

Relationships between cytosolic free calcium ([Ca2+]i) in platelets, indices of systemic calcium metabolism and blood pressure were examined in 86 subjects; 29 patients with untreated and 29 patients with treated essential hypertension, six patients with borderline hypertension and 22 healthy reference subjects. In order to analyse interactions between the variables, multivariate statistical analyses were employed. The patients with untreated hypertension had higher [Ca2+]i values in non-activated platelets (P = 0.04) and lower levels of plasma ionized calcium (P = 0.02) than the reference subjects. In multivariate models analysing platelet [Ca2+]i mean blood pressure (MBP), plasma ionized calcium, serum parathyroid hormone (PTH) and body mass index (BMI), the relationship between platelet [Ca2+]i and blood pressure was attenuated (P = 0.13), whereas the inverse relationships between plasma ionized calcium and MBP (P = 0.01) and between platelet [Ca2+]i and serum PTH (P = 0.06) seen in univariate analyses persisted. According to the multivariate models the [Ca2+]i value explained only 5% of the MBP variability. Thus, the data from this investigation do not support a close relationship between basal platelet [Ca2+]i and blood pressure. The inverse relationship between plasma ionized calcium and blood pressure, independent of platelet [Ca2+]i and serum PTH, suggests a direct interaction between plasma ionized calcium and blood pressure regulation.     

Platelet activation and subsequent inhibition by plasmin and recombinant tissue-type plasminogen activator.

The success of plasminogen activators in recanalizing occluded coronary arteries may be influenced by their effect on blood platelets; however, some previous studies have shown platelet activation by plasmin and thrombolytic agents while others have shown an inhibitory effect. Moreover, it has not been determined whether these effects reflect an alteration of intracellular signal transduction, fibrinogenolysis, degradation of adhesive protein receptors, or a combination of these events. To distinguish among these possibilities, the increase of cytoplasmic [Ca2+] [( Ca2+]i), which is an intracellular marker of platelet activation that precedes fibrinogen binding to the surface of activated platelets, was measured along with aggregation and release of 5-hydroxytryptamine (5-HT) in washed human platelets incubated with plasmin or recombinant tissue-type plasminogen activator (rt-PA). Plasmin (0.1 to 1.0 CU/mL) induced a prompt, concentration-dependent [Ca2+]i increase when added to platelets, but subsequently inhibited the [Ca2+]i increase in response to thrombin or the endoperoxide analog U44069. Platelet aggregation accompanied the [Ca2+]i increase if the platelets were stirred, while the aggregation of platelets unstirred during plasmin incubation was inhibited upon agonist addition and resumption of stirring. The release of 5-HT paralleled the [Ca2+]i increase induced by plasmin and was also inhibited after the subsequent addition of a second agonist. The effects of rt-PA, added with plasminogen (100 micrograms/mL), were similar to those of plasmin, and could be accounted for by the concentration of plasmin generated. The ADP scavengers apyrase and CP/CK each prevented the [Ca2+]i increase, and aggregation caused by plasmin or rt-PA, and also prevented their inhibitory effects on thrombin-induced activation. Thus, plasmin and rt-PA initially activate platelets, inducing a [Ca2+]i increase, and, if the platelets are stirred, aggregation. Such activation is followed by subsequent inhibition of cellular activation by a second agonist; the inhibitory effect is in proportion to the degree of initial activation, and ADP is an important cofactor in both processes. These platelet effects occur at rt-PA concentrations achievable clinically, and may affect the success of therapy with thrombolytic and adjunctive agents.     

Increased basal and thrombin-induced free calcium in platelets of essential hypertensive patients

Intracellular free calcium, [Ca2+]i, was studied in platelets of essential hypertensive subjects and normotensive controls under basal conditions and after stimulation with epinephrine, norepinephrine, angiotensin II, ouabain, and thrombin, using the fluorescent calcium indicator quin 2. Basal [Ca2+]i was significantly higher in hypertensive subjects (n = 32) than in normotensive controls (n = 30; 167.4 +/- 5.0 vs 143.2 +/- 3.1 nmol/L; p less than 0.001). Epinephrine, norepinephrine, angiotensin II, and ouabain had no effect on platelet calcium, whereas thrombin induced a dose-dependent increase in [Ca2+]i in both the presence and absence of extracellular calcium. This [Ca2+]i increase in the presence of extracellular calcium, which depends mainly on calcium influx, was significantly higher (p less than 0.05) in platelets of hypertensive subjects at all thrombin concentrations (ranging from 0.025-0.1 U/ml), while the [Ca2+]i increase in the absence of extracellular calcium, which depends only on release from intracellular stores, was similar in hypertensive subjects and controls. These results suggest that, in essential hypertension, there is not only increased platelet resting [Ca2+]i but also an increase in agonist-mediated calcium influx, which appears to indicate a cell membrane abnormality in the platelets of subjects with essential hypertension.     

Alpha 2-adrenergic inhibition of Cl- transport by opercular epithelium is mediated by intracellular Ca2+.

We isolated the opercular epithelium of sea-water killifish (Fundulus heteroclitus) to study the mediation of catecholamine inhibition of Cl- secretion. The receptors are alpha 2-adrenergic, as they have a high affinity for the alpha 2-adrenergic agonist clonidine over phenylephrine and clonidine action is blocked by yohimbine. Pertussis toxin and indomethacin did not block the clonidine effect; hence inhibitory guanine nucleotide-binding proteins (Gi proteins) and prostaglandins (respectively) are not involved. Intracellular pH (pHi) of single chloride cells was measured microspectrofluorometrically and resting pHi was 7.22 +/- 0.03. However, pHi was unaffected by clonidine; hence pHi and Na+/H+ exchange are not involved. The lipoxygenase inhibitors nordihydroguaiaretic acid and baicalein and the lipoxygenase products (12S)- and (12R)-12-hydroxyeicosatetraenoic acid stimulated Cl- secretion. Protein kinase C is an unlikely site of action because the diacylglycerol kinase inhibitor R59022 had no effect alone and did not block the clonidine effect. Ionomycin (1 microM) in normal but not low-Ca2+ solutions mimicked the action of clonidine and both inhibitions were reversible by isoproterenol. Thapsigargin, a releaser of intracellular Ca2+, inhibited Cl- secretion and this effect was reduced in low-Ca2+ solutions. Low-Ca2+ solutions also blunted but did not block entirely the clonidine response, indicating that the primary Ca2+ release was from intracellular stores. Whereas alpha 1-adrenergic receptors commonly act via the Ca2+/inositol trisphosphate pathway, to our knowledge this is the first report of a Ca(2+)-mediated alpha 2-adrenergic response in a nonmammalian vertebrate.     

Shear stress-induced von Willebrand factor binding to platelet glycoprotein Ib initiates calcium influx associated with aggregation.

Platelets subjected to elevated levels of fluid shear stress in the absence of exogenous agonists will aggregate. Shear stress-induced aggregation requires von Willebrand factor (vWF) multimers, extracellular calcium (Ca2+), adenosine diphosphate (ADP), and platelet membrane glycoprotein (GP)Ib and GPIIb-IIIa. The sequence of interaction of vWF multimers with platelet surface receptors and the effect of these interactions on platelet activation have not been determined. To elucidate the mechanism of shear stress-induced platelet aggregation, suspensions of washed platelets were subjected to different levels of uniform shear stress (15 to 120 dyne/cm2) in an optically modified cone and plate viscometer. Cytoplasmic ionized calcium ([Ca2+]i) and aggregation of platelets were monitored simultaneously during the application of shear stress; [Ca2+]i was measured using indo-1 loaded platelets and aggregation was measured as changes in light transmission. Basal [Ca2+]i was approximately 60 to 100 nmol/L. An increase of [Ca2+]i (up to greater than 1,000 nmol/L) was accompanied by synchronous aggregation, and both responses were dependent on the shear force and the presence of vWF multimers. EGTA chelation of extracellular Ca2+ completely inhibited vWF-mediated [Ca2+]i and aggregation responses to shear stress. Aurin tricarboxylic acid, which blocks the GPIb recognition site on the vWF monomer, and 6D1, a monoclonal antibody to GPIb, also completely inhibited platelet responses to shear stress. The tetrapeptide RGDS and the monoclonal antibody 10E5, which inhibit vWF binding to GPIIb-IIIa, partially inhibited shear stress-induced [Ca2+]i and aggregation responses. The combination of creatine phosphate/creatine phosphokinase, which converts ADP to adenosine triphosphate and blocks the effect of ADP released from stimulated platelets, inhibited shear stress-induced platelet aggregation without affecting the increase of [Ca2+]i. Neither the [Ca2+]i nor aggregation response to shear stress was inhibited by blocking platelet cyclooxygenase metabolism with acetylsalicylic acid. These results indicate that GPIb and extracellular Ca2+ are absolutely required for vWF-mediated [Ca2+]i and aggregation responses to imposed shear stress, and that the interaction of vWF multimers with GPIIb-IIIa potentiates these responses. Shear stress-induced elevation of platelet [Ca2+]i, but not aggregation, is independent of the effects of release ADP, and both responses occur independently of platelet cyclooxygenase metabolism. These results suggest that shear stress induces the binding of vWF multimers to platelet GPIb and this vWF-GPIb interaction causes an increase of [Ca2+]i and platelet aggregation, both of which are potentiated by vWF binding to the platelet GPIIb-IIIa complex.     

Changes in cytosolic calcium concentrations and cell morphology in single platelets adhered to fibrinogen-coated surface under flow

Changes in intracellular calcium concentration [Ca2+]i of fura-2-loaded human platelet during its adhesion to a fibrinogen-coated surface were studied, using a flow chamber mounted on an epifluorescence microscope equipped with digital-ratio imaging. Adherent platelets were individually mapped under a scanning electron microscope to establish the possible correlation between adhesion-associated shape alterations and [Ca2+]i changes. We found that 1) there was no immediate [Ca2+]i elevation on platelet adhesion; 2) [Ca2+]i changes varied drastically platelets with a lag time ranging 10 to 200 s, averaging about 1 minute; 3) the pattern of [Ca2+]i changes varied drastically among individual adherent platelets; 4) the degree of [Ca2+]i elevation appeared to correlate with the extent of morphology change, with the vast majority ( > 90%) of spread platelets showed detectable [Ca2+]i changes; 5) neither morphological nor [Ca2+]i changes correlated with the lag time; 6) platelets treated with dimethyl-BAPTA (15 mumol/L) underwent normal shape change without [Ca2+]i elevation; 7) cytochalasin D (10 mumol/L) inhibited both shape change and [Ca2+]i elevation; 8) colchicine (1 mmol/L) was ineffective in both regards. We conclude that although platelet adhesion-associated shape changes may be accompanied with heterogeneous [Ca2+]i changes that are microfilament-dependent, [Ca2+]i changes do not happen immediately after platelet-surface contact and they are not required for adherent platelets to undergo postcontact morphological changes.     

Alpha 1-adrenergic potentiation of vasoactive intestinal peptide stimulation of rat pinealocyte adenosine 3',5'-monophosphate and guanosine 3',5'-monophosphate: evidence for a role of calcium and protein kinase-C

alpha 1-Adrenergic agonists have recently been found to potentiate vasoactive intestinal peptide (VIP) stimulation of rat pinealocyte cAMP and cGMP. alpha 1-Adrenergic agonists also elevate pineal intracellular Ca2+ [( Ca2+]i) and activate protein kinase-C. In the present study, the possible involvement of Ca2+ and protein kinase-C in the alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP accumulation was examined with agents that alter [Ca2+]i or activate protein kinase-C. It was found that treatment with a Ca2+ chelator or with inorganic Ca2+ channel blockers inhibited alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP responses. Increasing [Ca2+]i by treatment with A23187, ouabain, or K+ potentiated VIP stimulation of cAMP and cGMP response. These observations indicate that Ca2+ mediates the alpha 1-adrenergic potentiation of VIP-stimulated cAMP and cGMP accumulation, as is true for the alpha 1-adrenergic potentiation of beta-adrenergic stimulated cAMP and cGMP accumulation. Activators of protein kinase-C mimicked the large effect alpha 1-adrenergic agonists have on cAMP accumulation in VIP-treated pinealocytes and had a small effect on cGMP accumulation in VIP-treated cells. These effects were not blocked by the Ca2+ chelator EGTA. However, the effects of a protein kinase-C activator on the cGMP response in VIP-stimulated cells were amplified by K+ (15 mM) or ouabain (1 microM), presumably through an action causing an increase in [Ca2+]i. These results suggest protein kinase-C is involved in the alpha 1-adrenergic potentiation of VIP-stimulated cAMP accumulation, as is the case for the alpha 1-adrenergic potentiation of beta-adrenergic stimulated cAMP. Protein kinase-C is also involved in cGMP accumulation, provided that there is a modest increase in [Ca2+]i.     

Alpha 1-adrenergic stimulation of in vitro growth hormone release and cytosolic free Ca2+ in rat somatotrophs

The effects of alpha 1-adrenergic agents on GH release and intracellular free Ca2+ concentration ([Ca2+]i) were investigated in purified rat somatotroph preparations. Phenylephrine (PHE) stimulated in vitro GH release; the maximal effect (2.5-fold stimulation) occurred at 1 microM PHE. The effect was completely blocked by the alpha-adrenergic antagonist phentolamine and partially counteracted by the beta-antagonist propranolol. Experiments with the fluorescent Ca2+ probe fura 2 show that PHE causes [Ca2+]i to rise from 178 +/- 31 nM (mean +/- SE; n = 25) to 370 +/- 55 nM (n = 9). This effect was complete within 20 sec and was maintained for at least 5-10 min. The rise was rapidly interrupted by administration of 1 microM phentolamine. The beta-receptor agonist isoproterenol caused a small [Ca2+]i rise due to action on alpha 1-adrenoreceptors. The PHE-induced [Ca2+]i rise showed two components: an initial peak due to Ca2+ mobilization from intracellular stores and a subsequent rise due to Ca2+ influx from the extracellular space. Somatostatin (SRIF) lowered both resting [Ca2+]i and Ca2+ influx stimulated by PHE. Pertussis toxin pretreatment did not modify PHE-induced [Ca2+]i changes, while it completely prevented the effect of SRIF on both resting and triggered [Ca2+]i, thus suggesting that a GTP-binding protein sensitive to the toxin is involved in the transduction of SRIF action. The increase in cAMP induced by cholera toxin pretreatment modified neither PHE nor SRIF action on [Ca2+]i. In conclusion, in rat somatotrophs Ca2+ mobilization and influx are stimulated by alpha 1-adrenergic agents, and this triggered [Ca2+]i rise results in a stimulation of GH release. In these cells SRIF is able to reduce both resting [Ca2+]i levels and [Ca2+]i increases induced by alpha 1-adrenergic activation.     

Human platelet calcium measurements. Methodological considerations and comparisons with calcium mobilization in vascular smooth muscle cells

Platelets are used as models for vascular smooth muscle cells (VSMC) in evaluating intracellular calcium ([Ca2+]i) metabolism in humans. This study was designed to determine if agonist-induced increases in [Ca2+]i in platelets occur via release from intracellular stores as previously demonstrated for VSMC. Incubation of purified platelets loaded with fura-2-AM in media containing 1.5 mmol/L Ca2+ resulted in higher basal [Ca2+]i than platelets incubated in Ca(2+)-free media. In addition, vasopressin-induced platelet [Ca2+]i transients were almost completely blocked by Ca2+ channel blockers. Thus, in contrast to VSMC, the transmembranous flux of extracellular Ca2+ is the major mechanism in vasopressin-induced increases in platelet [Ca2+]i, while mobilization of intracellular Ca2+ stores is only minimally involved.     

Epinephrine induces platelet fibrinogen receptor expression, fibrinogen binding, and aggregation in whole blood in the absence of other excitatory agonists

The exposure of fibrinogen receptors is an early event in agonist-induced platelet activation. Previous measurements of fibrinogen binding or aggregation in platelet-rich plasma or washed platelets have failed to define whether the initial response to epinephrine results solely from a direct effect of this agonist. To address this problem, we have measured fibrinogen receptor exposure on platelets in whole blood by using flow cytometry and a fluorescein isothiocyanate-labeled monoclonal antibody specific for the activated fibrinogen receptor (FITC-PAC1). We also measured platelet-bound fibrinogen with an antifibrinogen monoclonal antibody (FITC-9F9) as well as platelet aggregation in whole blood. In blood anticoagulated with citrate and in the presence of a cyclooxygenase inhibitor, epinephrine (0.1 to 100 mumol/L) caused significant FITC-PAC1 binding (P less than .001) that was maximal at 10 mumol/L epinephrine. The maximal epinephrine response was one third of that observed with 10 mumol/L adenosine diphosphate (ADP) and was eliminated by yohimbine, an alpha 2-adrenergic antagonist. Incubation of the blood with apyrase or phosphoenolpyruvate plus pyruvate kinase to remove extracellular ADP resulted in a 40% to 50% reduction in the epinephrine response. Despite this, FITC-PAC1 binding was still significant at epinephrine greater than or equal to 1 mumol/L (P less than .05). No reduction in epinephrine-induced FITC-PAC1 binding was observed in the presence of ATP alpha S, an ADP receptor antagonist; cinanserin, a serotonin antagonist; or WEB-2086, a platelet activating factor antagonist. Furthermore, addition of the thrombin inhibitors hirudin or leupeptin to citrated blood had no effect on the extent of the epinephrine response. Blood anticoagulated with hirudin also demonstrated an epinephrine response, even in the presence of apyrase. Similar results were obtained when FITC-9F9 was used to detect fibrinogen binding or when aggregation was assessed by a decrease in the number of single platelets. We conclude that epinephrine itself can induce fibrinogen receptor exposure, fibrinogen binding, and aggregation. This primary response is independent of synergistic interaction of epinephrine with traces of ADP, serotonin, platelet activating factor, or thrombin. However, such synergistic interaction with ADP present in whole blood may enhance the responses induced by epinephrine.     

Activation of protein kinase C in platelets by epinephrine and A23187: correlation with fibrinogen binding

Activation of protein kinase C (PKC), as revealed by phosphorylation of a 47 kd protein (p47), occurs in platelets stimulated by some agonists (eg, thrombin or phorbol esters). It is not known if activation of PKC occurs with pairs of agonists, such as epinephrine and A23187, that do not individually phosphorylate p47, nor is it known what role the concentration of cytoplasmic Ca++ ([Ca++]i) plays in these events. We stimulated aequorin-loaded platelets with subaggregating concentrations of epinephrine and A23187, neither of which by itself phosphorylated p47. The combination of agonists resulted in p47 phosphorylation, an increase in platelet-bound fibrinogen, and aggregation, but only if the concentration of each agonist was sufficient to increase [Ca++]i if it was added separately. Subaggregating concentrations of A23187 alone released platelet fibrinogen and increased platelet membrane binding of [3H]-phorbol dibutyrate, but these were not enhanced by epinephrine. Epinephrine and A23187 did not increase production of diacylglycerol. Thus, epinephrine and A23187 together activate PKC by a mechanism that does not require phospholipase C or enhanced binding of PKC to the plasma membrane; PKC activation in turn is correlated with enhanced platelet fibrinogen binding and aggregation. These events require an initial elevation of [Ca++]i above a threshold.     

Effect of systemic alpha-2 adrenergic blockade on the morning increase in platelet aggregation in normal subjects.

To assess whether activation of platelets by catecholamines contributes to the increase in platelet aggregation associated with either the assumption of upright posture or exercise, we studied the effect of oral yohimbine on these phenomena. Whole blood platelet aggregation and plasma catecholamine levels were measured before and after standing and at peak exercise in untreated normal subjects and after oral yohimbine. Neurochemical indexes indicated systemic alpha2-receptor blockade by yohimbine. Yohimbine reduced the orthostatic increase in platelet aggregation response by 63+/-11%, but exercise-induced increase in aggregation was unaffected. Thus, alpha2-adrenergic blockade attenuates the orthostatic increase in platelet aggregation. Agents designed to inhibit the morning surge in catecholamine levels or block platelet alpha2 adrenoceptors may reduce the risk of thrombotic vascular events in atherosclerosis.