Phosphoinositol Hydrolysis Research Articles

Progesterone attenuation of alpha 1-adrenergic receptor stimulation of phosphoinositol hydrolysis in hypothalamus of estrogen-primed female rats

These experiments examined whether the previously observed abolition by progesterone (P) of alpha 1-adrenergic potentiation of adenylyl cyclase activity in brain slices of estrogen-primed female rats is attributable to a reduced capacity of alpha 1-adrenoceptors to stimulate phosphoinositol hydrolysis. In preoptic area and hypothalamic slices from ovariectomized (OVX) female rats, both norepinephrine (NE) and the alpha 1-adrenergic agonist phenylephrine (PHE) were robust stimulators of inositol phosphate (IP) formation. The NE response was completely blocked by the alpha 1-adrenergic antagonist prazosin. PHE-induced IP formation in tissue from OVX females exposed for 48 h to estrogen alone was comparable to that in OVX controls. In hypothalamic tissue from OVX rats given estrogen plus P, NE and PHE activation of phosphoinositol hydrolysis was attenuated. Both chlorethylclonidine, an irreversible antagonist of alpha 1b-adrenoceptors, and 5-methylurapidil, an alpha 1a-selective antagonist, reduced NE-induced IP formation regardless of the hormonal condition of the animals. Analysis of PHE competition for [3H]prazosin binding indicated that agonist binding was of high affinity (Ki, 16-30 microM) and was unaffected by hormonal status. Therefore, P abolition of alpha 1-adrenergic augmentation of adenylyl cyclase is correlated with reduced phosphoinositol hydrolysis, but not with changes in alpha 1-adrenoceptor agonist binding affinity. Moreover, both alpha 1a- and alpha 1b-adrenoceptors appear to work together to stimulate this second messenger system in the female rat hypothalamus.

Progesterone attenuation of alpha 1-adrenergic receptor stimulation of phosphoinositol hydrolysis in hypothalamus of estrogen-primed female rats.

These experiments examined whether the previously observed abolition by progesterone (P) of alpha 1-adrenergic potentiation of adenylyl cyclase activity in brain slices of estrogen-primed female rats is attributable to a reduced capacity of alpha 1-adrenoceptors to stimulate phosphoinositol hydrolysis. In preoptic area and hypothalamic slices from ovariectomized (OVX) female rats, both norepinephrine (NE) and the alpha 1-adrenergic agonist phenylephrine (PHE) were robust stimulators of inositol phosphate (IP) formation. The NE response was completely blocked by the alpha 1-adrenergic antagonist prazosin. PHE-induced IP formation in tissue from OVX females exposed for 48 h to estrogen alone was comparable to that in OVX controls. In hypothalamic tissue from OVX rats given estrogen plus P, NE and PHE activation of phosphoinositol hydrolysis was attenuated. Both chlorethylclonidine, an irreversible antagonist of alpha 1b-adrenoceptors, and 5-methylurapidil, an alpha 1a-selective antagonist, reduced NE-induced IP formation regardless of the hormonal condition of the animals. Analysis of PHE competition for [3H]prazosin binding indicated that agonist binding was of high affinity (Ki, 16-30 microM) and was unaffected by hormonal status. Therefore, P abolition of alpha 1-adrenergic augmentation of adenylyl cyclase is correlated with reduced phosphoinositol hydrolysis, but not with changes in alpha 1-adrenoceptor agonist binding affinity. Moreover, both alpha 1a- and alpha 1b-adrenoceptors appear to work together to stimulate this second messenger system in the female rat hypothalamus.

Cellular expression of the carboxyl terminus of a G protein-coupled receptor kinase attenuates G beta gamma-mediated signaling.

The beta gamma subunits (G beta gamma) of heterotrimeric G proteins modulate the activity of several signal-transducing effector molecules including G protein-coupled receptor kinases. G beta gamma binds to the carboxyl terminus of the beta-adrenergic receptor kinase (beta ARK) and regulates its activity. To investigate the effect of such a G beta gamma-binding domain on heterologous G beta gamma interactions, various receptors that can stimulate phospholipase C and/or type II adenylate cyclase were coexpressed in COS-7 cells with the carboxyl terminus of beta ARK1. Phosphoinositol hydrolysis in response to activation of receptors that stimulate phospholipase C via Gi beta gamma (alpha 2-adrenergic and M2-muscarinic cholinergic receptors) was markedly inhibited by the coexpressed beta ARK1 polypeptide, whereas that mediated by Gq alpha subunits (alpha 1-adrenergic and M1-muscarinic cholinergic receptors) was unaffected. Increased cellular cAMP levels due to stimulation of receptors and coexpressed adenylate cyclase II displayed marked inhibition in the presence of the beta ARK1 polypeptide. Moreover, inhibition of adenylate cyclase produced by alpha 2-adrenergic receptor stimulation (a Gi alpha-mediated process) was unaffected, indicating that the beta ARK1 polypeptide provides a useful tool for distinguishing between G alpha and G beta gamma pathways.

Serines and threonines in the gastrin-releasing peptide receptor carboxyl terminus mediate internalization.

Most seven-transmembrane G-protein-coupled receptors are rapidly internalized after binding agonist, but the general amino acid recognition sequences mediating this phenomenon have not been identified. In this study, components of the gastrin-releasing peptide receptor (GRP-R) regulating internalization were identified. Four GRP-R mutants with stop codons placed at variable distances distal to the putative palmitoylation sites Cys340-341 were transiently expressed in CHOP fibroblasts. A construct with a minimal carboxyl tail deletion, T375, bound and internalized agonist similarly to wild type receptor. Progressively larger truncations of the carboxyl terminus, however, increasingly impaired GRP-R-mediated internalization without altering receptor-agonist affinity. Three additional constructs were created: one with the putative palmitoylation sites replaced with Ala (CC340-341AA), one with the carboxyl-terminal protein kinase C-consensus sequence converted to Ala (TS360-361AA), and one with all Ser and Thr distal to Cys341 converted to Ala, Asn, or Gly (JF1). All constructs bound agonist similarly to wild type receptor. CC340-341AA internalized similarly to native receptor (93 +/- 3% of wild type by 60 min), whereas internalization of TS360-361AA was partially attenuated (64 +/- 2% of wild type by 60 min). JF1, however, internalized as poorly as T346, with only 16 +/- 2% of the wild type receptors internalized by 60 min. To assess G-protein coupling, selected receptor constructs were stably transfected into Balb fibroblasts, and phosphoinositol hydrolysis was determined. The largest GRP-R truncation, T346, increased total inositol phosphates (EC50 = 2.9 +/- 0.9 nM) similarly to wild type receptor (EC50 = 5.1 +/- 2.2 nM), as did CC340-341AA (EC50 = 5.4 +/- 1.5 nM) and TS360-361AA (EC50 = 3.1 +/- 1.2 nM). These data demonstrate that the multiple Ser and Thr located within the GRP-R carboxyl terminus distal to Cys341, including but not limited to those within the protein kinase C-consensus sequence, specifically regulate GRP-R internalization rates independent of receptor-G-protein coupling.

The diurnal rhythm in vasopressin V1a receptor expression in the suprachiasmatic nucleus is not dependent on vasopressin.

The mammalian suprachiasmatic nucleus is the site of the circadian rhythm generator. The degrees of expression there of several neuropeptides, including vasopressin and vasoactive intestinal polypeptide, follow a diurnal rhythm. The vasopressin V1a receptor, whose activation results in phosphoinositol hydrolysis and mobilization of intracellular calcium, is expressed in this nucleus. This study used double simultaneous hybridization histochemistry to show that V1a receptor transcripts are present in both vasopressin and vasoactive intestinal polypeptide neurons and that their levels follow a diurnal rhythm. Furthermore, the expression of the V1a receptor is 12 h out of phase from that of vasopressin. However, the receptor's diurnal rhythm is still maintained in the vasopressin-deficient Brattleboro rat, indicating that the V1a receptor's rhythm is independent of any vasopressin feedback.

Oxytocin Pretreatment Enhances Arginine Vasopressin‐lnduced Motor Disturbances and Arginine Vasopressin‐lnduced Phosphoinositol Hydrolysis in Rat Septum: A Cross‐Sensitization Phenomenon

The recent observation that the central oxytocin (OT) receptor has high affinity for both OT and arginine vasopressin (AVP) raises the possibility that it may be involved in some of the central actions of AVP. Repeated intracerebroventricular (icv) injections of AVP in rats evoke an unusual sensitization phenomenon in that a first exposure to the peptide enhances the sensitivity (sensitization) of the brain to a second exposure. This report investigates the possibility that the OT receptor may be involved in the mediation of the phenomenon of sensitization, using OT, a specific OT receptor agonist, [Thr4,Gly7]OT, and a specific OT receptor antagonist, d(CH2)5,[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT (compound 6; cpd 6), as well as a V1 AVP receptor antagonist, d(CH2)5Tyr(Me)AVP. Peptides were injected icv in conscious, adult male Sprague-Dawley rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Protein kinase C activators stimulate betaendorphin secretion from hypothalamic cells

Relatively little is known about the regulation of secretion of hypothalamic beta-endorphin, the potent opioid that is believed to play a variety of physiological roles in brain. Previous work has shown that arginine vasopressin (AVP), which acts in brain primarily via activation of the phosphoinositol (PI) second messenger system, stimulates secretion of hypothalamic beta-endorphin. To test the hypothesis that activators of protein kinase C (PKC), which is activated following PI hydrolysis, stimulates secretion of beta-endorphins from hypothalamus, we studied the separate effects of stimulators of PKC including phorbol ester 12-myristate-13-acetate (PMA) and 1-oleolyl-2-acetyl glycerol (OAG — a diacyl glycerol analogue) on secretion of immunoreactive (IR-) beta-endorphin (measured by RIA) from dissociated fetal rat hypothalamic cell cultures. We also studied AVP and angiotensin II (Ang II), hypothalamic peptides which activate the PI second messenger pathway, and interactions of PMA and forskolin (FSK), an activator of the cyclic AMP/protein kinase A (PKA) pathway. PMA, OAG, AVP, and Ang II stimulated IR-beta-endorphin secretion. The stimulatory effect of both PMA and FSK on IR-beta-endorphin secretion was greater than that of PMA or FSK alone and was essentially additive. From these results we conclude that 1. (a) hypothalamic beta-endorphin secretion can be stimulated by activating PKC; 2. (b) AVP and Ang II are potential physiological regulators of hypothalamic beta-endorphin secretion; 3. (c) coupling of PKC activation and stimulation of hypothalamic IR-beta-endorphin secretion which is presumed at maturity (adulthood) originates during early development (fetal life); 4. (d) stimulating hypothalamic beta-endorphin secretion via PKC activation utilizes a distinct, different mechanism than stimulation of hypothalamic beta-endorphin secretion via PKA activation.

U-73122, an aminosteroid phospholipase C antagonist, noncompetitively inhibits thyrotropin-releasing hormone effects in GH3 rat pituitary cells.

TRH increases cytosolic-free calcium ([Ca2+]i) by activating phospholipase C(PL-C), which induces phosphoinositol hydrolysis, leading to Ca2+ mobilization from inositol trisphosphate (IP3) sensitive stores, and by increasing Ca2+ influx. Increases in [Ca2+]i stimulate PRL secretion. We investigated the effects of U-73122, an aminosteroid inhibitor of PL-C dependent processes, on TRH-stimulated second messenger pathways and on PRL secretion in GH3 rat pituitary cells. [Ca2+]i was monitored by Indo-1 fluorescence, and IP3 and metabolites separated on ion exchange columns. In Ca(2+)-free buffer, [Ca2+]i was 96 +/- 6 nM and increased to 323 +/- 23 nM (P less than 0.001) after TRH (100 nM). U-73122 dose dependently inhibited the TRH effect (IC50 = 967 nM; complete inhibition at 3-5 microM). Subsequent addition of monensin (100 microM) increased [Ca2+]i from 107 +/- 4 to 142 +/- 4 nM (P < 0.001), confirming our previous findings of a non-TRH regulated Ca2+ pool in GH3 cells. Pretreatment (15 sec) with U-73122 partly inhibited the TRH effect on [Ca2+]i; complete suppression occurred with 70 sec of pretreatment. An inactive analog (U-73343) had no inhibitory effect at 5 microM. U-73122 acted noncompetitively, as the mean maximum velocity (expressed as percent increase in [Ca2+]i after TRH) was reduced from 225 to 91 while the Michaelis-Menten constant for TRH was unchanged (15.4 vs. 13.8 nM, n = 3). Of note, U-73122, at 3-5 microM, increased basal [Ca2+]i from 109 +/- 5 to 120 +/- 5 nM (P less than 0.001). In 1.3 mM Ca2+ buffer containing nifedipine (1 microM) and verapamil (50 microM), similar effects of U-73122 (5 microM) were observed on basal and TRH-stimulated [Ca2+]i. IP3, IP2, and IP1 increased to 241 +/- 12%, 148 +/- 23%, and 167 +/- 39% of control, 30 sec after TRH (100 nM); these responses were prevented by 1 microM U-73122. At 5 microM, U-73122 also significantly increased IP3 levels. TRH (100 nM) increased 4-h PRL secretion from 16.3 +/- 1.4 to 27.6 +/- 3.2 ng/well (P less than 0.05). U-73122 (5 microM) increased basal PRL secretion to 35.9 +/- 3.2 ng/well (P less than 0.05), but abolished the TRH effect. In contrast, U-73343 (with Ca2+ channel blockers) did not inhibit the TRH effect on PRL (control: 24.3 +/- 2.1; TRH: 51.0 +/- 6.3 ng/well).(ABSTRACT TRUNCATED AT 400 WORDS)

Cellular physiology and pathophysiology of the parathyroid glands

This report provides insight into parathyroid gland physiology and the pathophysiology of hyperparathyroidism (HPT). Increases in the extracellular calcium concentration constitute the primary physiological signal for inhibition of parathyroid hormone (PTH) release. Transduction of the external signal into a cellular response involves activation of a cation receptor mechanism on the plasma membrane with rapid rise in the cytoplasmic calcium concentration of the cells. This recently discovered parathyroid calcium receptor has been characterized as a glycoprotein of unusually high molecular weight, which may play a key role in calcium homeostasis since it is also expressed in the kidney and placenta. Binding of external calcium to the receptor is associated with mobilization of intracellular calcium as well as calcium influx into the cells and phosphoinositol hydrolysis. These events rapidly interfere with the release process through essentially unknown mechanisms and probably also at sustained stimulation inhibit PTH gene transcription. The relative calcium insensitivity of the PTH release in HPT is associated with a deranged regulation of cytoplasmic calcium within pathological parathyroid cells. The molecular basis for this disturbance comprises down regulation of the cation receptor, whereby external calcium is translated into abnormally low levels of cytoplasmic calcium and insufficient inhibition of PTH release. Studies on expression of the functionally important cation sensing glycoprotein and its associated cellular signal systems may provide novel means for interference with the pathophysiological derangements of HPT.

Characterization of endothelin 1 receptor and signal transduction mechanisms in rat medullary interstitial cells

Previous autoradiographic studies have delineated the renal medullas the predominant site of renal endothelin (ET) receptors. Accordingly, cultured rat renal medullary interstitial cells (RMICs) were studied as a target tissue for ET action. Scatchard analysis revealed presence of a single class of high-affinity receptor sites (Kd, 57 +/- 10 pM; receptor density, 749 +/- 124 fmol/mg protein). Relative potency order for displacing 125I-ET-1 was ET-1 greater than ET-2 greater than sarafotoxin greater than big endothelin (human) = big endothelin (porcine). ET-3, unrelated pressor substances, vasodilators, Ca2+ channel antagonists, atrial natriuretic factor, GTP, and GppNHp did not inhibit binding. Challenge of monolayers with ET-1 evoked a biphasic elevation in cytosolic free Ca2+ concentration [Ca2+]i). Initial transient rise in [Ca2+]i observed in absence of extracellular Ca2+ and accumulation of inositol trisphosphate (IP3) was consistent with activation of phosphatidylinositol-specific phospholipase C (PI-PLC). Half-maximal activation concentration of ET-1 for the process was 0.5 and 1 nM for [Ca2+]i and IP3, respectively. The late sustained phase in [Ca2+]i elevation was completely blocked by Ni2+, unperturbed by nimodipine, and accompanied by influx of Mn2+, indicating presence of receptor-operated Ca2+ channels. Ca2+ channel opening was detected at 10(-16) MET-1, whereas greater than 10(-12) M agonist was required to mobilize Ca2+ from intracellular stores and/or stimulate phosphoinositol hydrolysis, indicating that ET activation of PI-PLC and Ca2+ channel opening were independent events. ET-1 markedly stimulated prostaglandin E2 synthesis in a concentration-dependent manner that paralleled PI-PLC activation and mobilization of [Ca2+]i. In summary, cultured rat RMICs possess ET receptors that are linked to PI-PLC, Ca2+ channels, and perhaps phospholipase A2.

Enzymatic formation and regulatory function of lipoxins and leukotriene B4 in rat kidney mesangial cells.

Many metabolites of arachidonic acid (AA) have been demonstrated to act as second messengers for cellular functions and signal transduction. Following liberation from membrane phospholipids by the actions of phospholipases, arachidonic acid (AA) can be further metabolized by cyclooxygenase (CO), lipoxygenase (LO), and epoxygenase enzyme systems to generate oxygenated metabolites of AA that participate in the regulation of cell function. There are at least three lipoxygenases responsible for the generation of biologically active metabolites from AA. These are the 5-, 12-and 15-lipoxygenases, which give rise to the predominately pro-inflammatory leukotrienes (LTs) and the mono and di-hydroxy eicosatetraenoic acids (HETEs). Recently lipoxins, a class of structurally related trihydroxytetraenes derived from AA and eicosapentaenoic acid (EPA) have been described (1, 2). These compounds are formed by the sequential oxidation of these polyunsaturated fatty acids by 5-lipoxygenase and 15-Lipoxygenase or 12-Lipoxygenase (3,4). Lipoxins possess potent biological activities which are distinct from those of other lipoxygenase and cyclooxygenase products (5) and in many respects lipoxin have an apparent anti-inflammatory function. For example, prior exposure of neutrophils to LXA4, inhibits LTB4 induced phosphoinositol hydrolysis and impairs the chemotic response of polymorphonuclear cells to both LTB4 and formyl-methionyl-leucyl-phenylalanine (fMLP) (6).

Central vasopressin pretreatment sensitizes phosphoinositol hydrolysis in the rat septum

Previous in vivo and in vitro studies have demonstrated that exposure of the brain to arginine vasopressin (AVP) can potentiate various responses to a second central challenge with AVP. To determine whether this sensitization is mediated by changes at the receptor level, we investigated the effects of AVP on the phosphoinositide metabolism in septal slices prepared from rats centrally pretreated with saline or AVP. Addition of vasopressin (10 −7 M, 10 −6 M) to septal slices from saline-pretreated rats failed to elicit a significant stimulation of inositol-1-phosphate (IP 1). In contrast, AVP (10 −7 M) significantly stimulated IP 1 release in septal slices prepared from rats pretreated intracerebroventricularly (i.c.v.) 24 h earlier with 10 or 100 ng AVP. Pretreatment with the same i.c.v. doses of AVP also induced a significant enhancement of the carbachol-induced stimulation of IP 1 release, but i.c.v. pretreatment with carbachol did not stimulate the IP 1 release in response to AVP. Our results suggest that a novel facilitation of phosphoinositide metabolism can be induced by central AVP pretreatment.

Protein kinase C and intestinal fluid secretion: involvement of prostaglandin E 2 but not of 5-hydroxytryptamine

To determine the role of protein kinase C in the regulation of intestinal fluid transport, experiments were performed with the rat jejunum in vivo, using the active phorbol ester, 4-β-phorbol 12-myristate 13-acetate (PMA), as stimulator of protein kinase C. Intraluminally administered PMA dose dependently reversed the net fluid absorption to net fluid secretion and significantly increased prostaglandin E 2 (PGE 2) but not 5-hydroxytryptamine (5-HT) output into the lumen. Mucosal cyclic AMP levels remained unchanged by PMA. Indomethacin inhibited the increase in PGE 2 output and partially reduced the secretory response to PMA. Ketanserin was without effect whereas verapamil totally blocked the secretory response to PMA. It is concluded that intestinal fluid secretion, stimulated by activation of protein kinase C is partly mediated by PGE 2 release. PGE 2 may facilitate calcium entry rather than increase intracellular calcium through activation of cyclic AMP. Protein kinase C appears to play an important role as an intermediate in phosphoinositol hydrolysis, which is initiated by 5-HT, and finally induces fluid secretion via PGE 2.

Gestational alterations in phospholipase C coupled muscarinic response

In the pregnant rat, carbachol-induced phosphoinositol hydrolysis by myometrium at the placental atatchment region prosressively decreased toward term, whereas hydrolysis was relatively stable in the myometrium of the non-attachment region. Tritium-quinuclidinyl benzilate binding increased in the myometrium of non-attachment regions as pregnancy progressed. At placental attachment sites binding remained relatively stable until parturition when it increased. Apparently the myometrium associated with the placental attachment site is less sensitive to cholinergic influence during pregnancy compared with the non-attachment site when evaluated by muscarinic activation of phospholipase C or ligand binding.

Enhanced phosphoinositol hydrolysis in response to vasopressin in the septum of the homozygous brattleboro rat

Arginine 8-vasopressin (AVP) receptors in the septum of the Long-Evans rat have been shown to be both pharmacologically (displacement profiles) and functionally (ability to stimulate phosphoinositide hydrolysis) similar to the peripheral V 1-type receptor for AVP. Previous binding studies of AVP receptors in the septum of heterozygous (HE) and homozygous (vasopressin-deficient, HO) Brattleboro (BB) rats revealed an increased number of receptors with a lower affinity for AVP in the HO-BB rat when compared to the HE-BB rat. To determine the effect of these receptor changes in the HO-BB rat septum on the postreceptor response of the tissue to AVP, concentration-response relationships for AVP-stimulated phosphoinositide hydrolysis were examined in septal slices from age-matched, adult male HE- and HO-BB rats. AVP-stimulated accumulation of [ 3H]inositol-1-phosphate (IP 1) was significantly greater in the HO-BB (43.7%) than in the HE-BB (13.7%) at AVP concentrations of 10 −08 to 10 −05 M. The two groups did not, however, differ in their ability to stimulate [ 3H]IP 1 accumulation in response to 2.0 nM carbochol. When the AVP-stimulated phosphoinositide response in both genotypes was compared to that obtained for the Long-Evans (LE) rat (the parent strain of the Brattleboro rat) septum under the same assay conditions, it was found that the response in the HE-BB was much lower in the LE, AVP receptor binding capacity ( B max correlated ( r = 0.975) with release of IP 1 [ 3H]P 1 accumulation) for all 3 groups studied (LE, HE, HO). These results indicate that Brattleboro rats do posses functional receptors for AVP in the septum, and that the postreceptor response in the septum of the HO-BB rat is much larger than in the HE-BB rat.

Release of Endothelium-Derived Relaxing Factor is Inhibited by 8-Bromo-Cyclic Guanosine Monophosphate

Endothelial cells are known to contain both soluble and particulate guanylate cyclase, but the functional role of cyclic guanosine monophosphate (cGMP) in endothelial cells remains unknown. We have investigated the effects of 8-bromo-cGMP on endothelium-dependent relaxations to acetylcholine, substance P, ATP, and the calcium ionophore A23187, and on endothelium-independent relaxations to sodium nitroprusside and glyceryl trinitrate (GTN). The ability of each of these agents to relax phenylephrine-preconstricted rings of rabbit aorta was tested in the absence and presence of 8-bromo-cGMP. In the presence of 8-bromo-cGMP, a greater concentration of phenylephrine had to be used to produce a similar level of tone and then endothelium-dependent relaxations to acetylcholine and substance P were inhibited, whereas endothelium-dependent relaxations to ATP and A23187 were unaffected. Endothelium-independent relaxations to sodium nitroprusside and GTN were only inhibited at the highest concentrations of nitroprusside and GTN. These results suggest that: (a) increasing GMP levels in endothelial cells inhibit agonist-induced release of endothelium-derived relaxing factor (EDRF); (b) a negative feedback mechanism may exist whereby EDRF stimulates soluble guanylate cyclase in endothelial cells to inhibit its own release; and (c) ATP does not induce EDRF release via phosphoinositol hydrolysis.

A pertussis toxin substrate regulates alpha 1-adrenergic dependent phosphatidylinositol hydrolysis in cultured rat myocytes.

The chronotropic response of the heart to alpha 1-adrenergic catecholamines influenced by pertussis toxin under certain conditions. In view of the fact that alpha 1-adrenergic action is mediated by the phosphatidylinositol pathway of hormone action in many cells, we examined the hypothesis that alpha-adrenergic agonists stimulate phosphatidylinositol hydrolysis in cardiomyocytes and that this effect is sensitive to pertussis toxin. Addition of norepinephrine to cultured rat ventricular myocytes prelabeled with myo-[2-3H]inositol resulted in rapid and significant accumulation of inositol phosphate (IP1) and inositol biphosphate. Norepinephrine-stimulated IP1 formation was not inhibited by propranolol, but was inhibited by alpha-adrenergic antagonists with an order of potency indicating alpha 1-adrenergic receptor subselectivity: prazosin (alpha 1; 3 nM) greater than yohimbine (alpha 2; 10 microM). The effect of norepinephrine to enhance IP1 formation was markedly attenuated in cells pretreated with pertussis toxin. Pertussis toxin also induced the transfer of ADP-ribose from NAD to a 41,000-dalton membrane protein in these cells. The concentration of pertussis toxin resulting in maximal inhibition of norepinephrine-stimulated IP1 formation correlated well with the concentration of pertussis toxin necessary to completely ADP-ribosylate a 41,000-dalton membrane protein (1 ng/ml). The range over which pertussis toxin inhibited norepinephrine-dependent IP1 formation and ADP-ribosylated the 41,000-dalton substrate was virtually identical. These observations establish a role for a 41,000-dalton pertussis toxin substrate in coupling the alpha 1-adrenergic receptor to phosphoinositol hydrolysis in myocardial cells.