Tetracaine blocks the responses of isolated acinar cells from rat parotid to carbachol but not to substance P.

Carbachol and substance P stimulated 45Ca2+ flux changes, 86Rb+ efflux, and amylase secretion from acinar cells isolated from rat parotid. The local anesthetic tetracaine blocked all of these measured responses to carbachol, but none of the responses to substance P. Tetracaine must act at either the cholinergic receptor or at a subsequent transducing step in the cholinergic stimulus-response sequence. If tetracaine acts at one of the transducing steps between cholinergic receptor occupation and the physiological responses then the action of tetracaine must be at a locus in the cholinergic reaction scheme not shared by substance P, because tetracaine did not block any response of the parotid to substance P.     

Specific binding of an immunoreactive and biologically active 125I-labeled N(1)acylated substance P derivative to parotid cells

A biologically active ¹²⁵I-substance P derivative (I¹²⁵-BH-substance P), prepared by conjugation of substance P with [^125I]Bolton-Hunter reagent, binds specifically to isolated rat parotid cells. The Kd is 4 nM for I-BH-substance P, 5 nM for substance P, 0.18 μM for substance P octa(4–11)peptide, and 1.6 μM for substance P [pyroglutamyl⁶]hexa(6–11)peptide. Substance P free acid and substance P penta(7–11)peptide are much weaker competitors and the C-terminal tri(9–11)peptide has no effect at 30 μM. The binding is also inhibited by 1 μM physalaemin, eledoisin and substance P methyl ester, but not by unrelated peptides. The selective inhibition of the binding by the biologically active analogs and fragments of substance P indicates that the ¹²⁵I-labeled N(1)acylated substance P derivative may interact with a substance P receptor on parotid cells.     

Effects of alkylating antagonists on the stimulated turnover of phosphatidylinositol produced by a variety of calcium-mobilising receptor systems

We have investigated the effects of two halogenoalkylamine drugs, dibenamine and phenoxybenzamine, on the stimulated phosphatidylinositol turnover that is produced by neurotransmitters and hormones which interact with receptors to bring about an increase in cell surface Ca²⁺ permeability. The phosphatidylinositol responses we have investigated were those evoked by muscarinic cholinergic stimuli (parotid gland and pancreas), by α-adrenergic stimuli (parotid gland, vas deferens smooth muscle), by pancreozymin or caerulein (pancreas), by phytohaemagglutinin (lymphocytes) and by either 5-hydroxytryptamine or elevation of the extracellular K⁺ concentration (ileum smooth muscle). Phenoxybenzamine inhibited the muscarinic cholinergic, α-adrenergic, 5-hydroxytryptamine and high K⁺ responses, but not the responses to phytohaemagglutinin and to pancreozymin (or caerulein). Dibenamine was less effective than phenoxybenzamine in inhibiting the α-adrenergic response and the high K⁺ response, and it did not inhibit the responses to muscarinic cholinergic stimuli, to 5-hydroxytryptamine or to the polypeptides. N,N-dimethyl-2-bromo-2-phenylethylamine (DMPEA) inhibited the α-adrenergic response, but not the response to muscarinic cholinergic stimulation. The specificity of DMPEA for the α-adrenergic response agrees with its postulated site of action at the noradrenaline-binding site of this receptor system, whereas dibenamine and phenoxybenzamine are less specific drugs which inhibit a variety of the ‘physiological’ responses of cells, including those to muscarinic cholinergic, H₁-histaminergic, α-adrenergic and 5-hydroxytryptamine stimuli. Previously, we suggested that dibenamine and phenoxybenzamine might show a constant pattern of effects on the phosphatidylinositol responses evoked through different receptors, phenoxybenzamine being inhibitory and dibenamine without effect [Jafferji & Michell (1976) Biochem. J. $\text{160}$, 163–169]. However, this pattern has not been sustained throughout the present study of a larger range of Ca²⁺-mobilising stimuli.     

Effect of substance P and eledoisin on K efflux, amylase release and cyclic nucleotide levels in slices of rat parotid gland

The undecapeptides, substance P and eledosin, caused a rapid, concentration-dependent increase in K⁺ efflux and amylase release from parotid tissue slices. The effects were not blocked by β-adrenergic, α-adrenergic, or cholinergic anatagonists. Incubation buffer calcium was required for stimulation of K⁺ efflux and amylase release. The action of the undecapeptides was independent of any effects on parotid cyclic AMP or cyclic GMP levels. Since the actions of the undecapeptides were Ca²⁺ dependent and no effects on cyclic nucleotide levels were discerned it was concluded that Ca²⁺ plays a primary role in agonist regulation of K⁺ efflux from the parotid.     

Crosstalk between purinergic receptors and canonical signaling pathways in the mouse salivary gland

Isolated clusters of mouse parotid acinar cells in combination with live cell imaging were used to explore the crosstalk in molecular signaling between purinergic, cholinergic and adrenergic pathways that integrate to control fluid and protein secretion. This crosstalk was manifested by (1) β-adrenergic receptor activation and amplification of P2X4R evoked Ca²⁺ signals, (2) β-adrenergic-induced amplification of P2X7R-evoked Ca²⁺ signals and (3) muscarinic receptor induced activation of P2X7Rs via exocytotic activity. The findings from our study reveal that purinoceptor-mediated Ca²⁺ signaling is modulated by crosstalk with canonical signaling pathways in parotid acinar cells. Integration of these signals are likely important for dynamic control of saliva secretion to match physiological demand in the parotid gland.     

Rapid desensitization of substance P- but not carbachol-induced increases in inositol trisphosphate and intracellular Ca++ in rat parotid acinar cells

The effects of supramaximal concentrations of substance P and the cholinergic agonist carbachol on the accumulation of inositol trisphosphate and the elevation of the intracellular free calcium concentration were compared in rat parotid acinar cells. Substance P was fully as effective as carbachol at initial times, but there was a rapid loss of the substance P responses while the effects of carbachol were well maintained. The loss of the substance P responses represented desensitization rather than degradation of the peptide since further additions of substance P were without effect. Desensitization to substance P did not involve long-term loss of substance P receptors as it was fully reversible in less than twenty minutes, the minimum time to extensively wash previously desensitized cells.     

Oxygen consumption in the parotid gland

Carbachol, substance P and epinephrine, but not isoproterenol, transiently stimulated O₂ consumption by 40–50% in rat parotid gland slices. The response to carbachol, but not to substance P, was blocked by atropine. Ouabain (1 mM) did not affect the response to carbachol. Also, the response to carbachol did not require external Ca, and was not significantly diminished by 1.0 mM LaCl₃. Reintroduction of Ca to a low Ca medium increased O₂ consumption only if carbachol was present. Procaine inhibited the increase in O₂ consumption due to carbachol, but not that due to substance P. When both carbachol and substance P were presented to the tissues in series and in the absence of external Ca, the second agonist failed to produce a response. When these results are considered in the light of previous studies on Ca and the responses of the parotid gland, they suggest that the primary stimulus for the O₂ consumption is the release of a limited pool of membrane-bound Ca following receptor activation.     

Substance P degrading systems of rat parotid and hypothalamus

Inactivation of substance P and its C-terminal hexapeptide analog [p-Glu⁶]substance P6–11 was studied in rat parotid and hypothalamic slices. It was found that in the parotid slice system the decay of substance P induced K⁺ release occurs concurrently with a decrease in the biologically active concentration of the peptide in the medium. The inactivation was further studied using [p-Glu⁶]substance P6–11 as substrate in the parotid and in the hypothalamic slice systems. In both tissue preparations the hexapeptide is degraded to small peptide fragments by metalloendopeptidase. Separation of the peptide fragments by high performance liquid chromatography and determination of their amino acid composition showed that in the hypothalamic slice system the major cleavage of the hexapeptide analog occurs between Phe⁸-Gly⁹ with minor cleavage sites between Phe⁷-Phe⁸ and Gly⁹-Leu¹⁰. In the rat parotid slice system the major cleavage occurs between Gly⁹-Leu¹⁰ with a minor cleavage site between Phe⁷-Phe⁸. The degradation of the hexapeptide analog in the hypothalamic system was inhibited 77% and 67% by treatment with 1 mM p-chloromercuriphenylsulfonate and p-chloromercuribenzoate, respectively, whereas in the parotid system these reagents inhibited the degradation of the hexapeptide only by 15% and 8%. These results may indicate that different proteases in the parotid and hypothalamus are involved in degradation of substance P. Kinetic studies, including the use of various inhibitors as well as competition by the peptide hormones somatostatin, LHRH, TRH and Leu-enkephalin-NH₂, revealed that in both tissues the hexapeptide analog is a preferred substrate for degradation by protease of considerable specificity towards the C-terminal sequence of substance P. It is suggested that this metalloendopeptidase may be important in the termination of the substance P response.     

cGMP production is coupled to Ca-dependent nitric oxide generation in rabbit parotid acinar cells

We investigated the mechanism of guanosine 3′,5′-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a β-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca²⁺ mobilization, because intracellular Ca ²⁺ elevating reagents, such as thapsigargin and the Ca²⁺ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca²⁺ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by N^G-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca²⁺. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca²⁺ mobilization.     

Supersensitivity to substance P after dorsal root section.

Two to three weeks after unilateral dorsal root section, specific [³H] substance P binding in synaptic membranes from rabbit spinal cord increased significantly compared with that in intact side, while non-specific [³H] substance P binding was unchanged. Scatchard analysis indicates that the development of such supersensitivity is due to an increase in a number of receptor sites, not to an increase in a receptor affinity. These results strongly support the notion that substance P exerts a transmitter role for primary afferent fibers.     

The modulatory effects of leukotrienes C4 and D4 on methacholine- and substance P-induced salivary secretion in rats

Although certain prostaglandins have been found to be inhibitory to nerve-evoked salivary flow, little is known of the effects the leukotrienes on salivary secretion. It was the purpose of this investigation to examine the effects of leukotrienes C₄ (LTC₄) and D₄ (LtD₄) on salivary secretion in the rat, using methacholine or substance P to induce basal secretion, and to test whether or not the observed effects of these eicosanoids were receptor-mediated by using the leukotriene receptor blocker FPL-55712.Methacholine (3 × 10⁻⁴ M), or substance P (1 × 10⁻⁶ M) was infused intra-arterially to stimulate secretion and saliva was collected separately from the parotid gland and the submandibular gland of anesthetized rats. LTC₄ and LTD₄ (each at 1 × 10⁻⁹ to 1 × 10⁻⁶ M) were found to reduce methacholine- and substance P-induced salivary flow in a dose-related manner. Salivary protein concentration and amylase activity were not significantly altered by the leukotrienes; however, arginine-esterase activity, stimulated by substance P, was increased by both leukotrienes. FPL-55712 (1 × 10⁻⁸ M) was shown to reduced the inhibitory effects of LTC₄ and LTD₄, suggesting the involvement of leukotriene receptors for these agents in their action.     

Low affinity purinergic receptor modulates the response of rat submandibular glands to carbachol and substance P

The effect of extracellular ATP on the intracellular calcium concentration ([Ca²⁺]_i) in rat submandibular glands was tested. The dose-response curve for ATP was biphasic with a first increase in the 1–30 μM concentration range and a further increase at concentrations higher than 100 μM. Among ATP analogs, only benzoyl-ATP stimulated the low affinity component. ATPτS blocked this response. All the other analogs tested reproduced the high-affinity low capacity response. Magnesium and Coomassie blue selectively blocked the low affinity component. High concentrations of ATP blocked the increase of the intracellular calcium concentration [Ca²⁺]_i in response to 100 μM carbachol. By itself, substance P (100 pM-1 μM) increased the [Ca²⁺]_i. One mM ATP potentiated the response to concentrations of substance P higher than 10 nM. This potentiation was reversed by extracellular magnesium. Carbachol 100 μM and substance P (100 pM-1 μM) increased the release of inositol trisphosphate (IP₃) from polyphosphoinositides (polyPI). Activation of the low affinity ATP receptors did not activate the polyPI-specific phospholipase C but inhibited its activation by 100 μM carbachol (−50%) and by 100 nM substance P (−60% at 1 nM substance P and −40% at 100 nM substance P). Substance P induced a strong homologous desensitization: a preincubation with 1 nM substance P nearly completely abolished the response to 1 μM substance P. When the cells were exposed to ATP before the second addition of substance P, the purinergic agonist partially restored the response to the tachykinin without totally reversing the desensitization. It is concluded that two types of purinergic receptors coexist in rat submandibular glands; a high-affinity, low capacity receptor which remains pharmacologically and functionally undefined and a low affinity site, high capacity receptor of the P_2Z type coupled to a non-selective cation channel. The occupancy of these low affinity sites blocks the increase of the [Ca²⁺]_i in response to a muscarinic agonist and the activation of polyPI-specific phospholipase C by carbachol and substance P. It potentiates the effect of high concentrations of substance P on the [Ca²⁺]_i. © 1996 Wiley-Liss, Inc.     

Activation by calcium of membrane channels for potassium in exocrine gland cells

In the rat parotid salivary gland, fluid secretion is regulated by alterations in fluxes of monovalent ions. , stimulation of muscarinic, α-adrenergic or substance P receptors provokes a biphasic increase in membrane permeability to K⁺ which can be conveniently assayed as efflux of ⁸⁶Rb. The increased ⁸⁶Rb flux is thought to arise in response to a receptor mediated elevation in [Ca²⁺]_i which activates Ca²⁺-activated K⁺-channels. The biphasic nature of the response is presumably due to a biphasic mode of Ca²⁺ mobilization by secretagogues; a transient response reflects release of a finite pool of Ca from an intracellular store while a more sustained phase results from Ca entry through receptor operated Ca channels or gates. Calcium also mediates an increased Na⁺ entry which in turn activates the Na⁺, K⁺-pump. The mechanism involved in the regulation of monovalent ion channels by Ca²⁺ is not understood.     

Effects of Substance P on the Binding of Agonists to the Nicotinic Acetylcholine Receptor of Torpedo Electroplaque

Abstract: Abstract: The effect of the neuropeptide substance P on the binding of the cholinergic ligands to the nicotinic acetylcholine receptor of Torpedo electroplaque membranes was examined at a physiological concentration of NaCl (150 m M ). Substance P had no effect on the initial rate of ¹²⁵I-α-bungarotoxin binding at concentrations of <100 μ M . The peptide did not bind to the high-affinity local anesthetic site but allosterically modulated [³H]phencyclidine binding, positively in the absence of agonist and negatively in the presence of agonist. Substance P increased the apparent affinity of the cholinergic agonists carbamylcholine and acetylcholine at equilibrium. The effect of substance P on the equilibrium binding of [³H]acetylcholine was examined directly, and the peptide appeared to increase the affinity of the binding of the second molecule of agonist, with no effect on the binding of the first. This indicates that substance P can affect the cooperative interactions between agonist binding sites. Substance P appeared to increase the rate of carbamylcholine-induced desensitization; however, the data are also consistent with an allosteric mechanism that does not involve the desensitized state. To attempt to differentiate between these mechanisms, the rates of recovery were determined after exposure to peptide and/or agonist. The kinetics of recovery are consistent with stabilization of the desensitized state by substance P if the peptide remains bound long enough to allow rapid recovery to the low-affinity state. However, an allosteric modulation of agonist binding that does not involve the desensitized state cannot be ruled out.     

Sphingosine inhibits muscarinic cholinergic receptor binding in rat parotid acinar cells

1. Sphingosine inhibited the binding of [³H]quinuclidinyl benzilate (QNB), a potent and specific muscarinic antagonist, in dispersed rat parotid acinar cells.2. The inhibition of [³H]QNB binding was expressed as decrease in affinity without significant change of a number of membrane sites.3. The effect of Sphingosine on the binding was not affected by the chelation of extracellular Ca²⁺.4. H-7, an inhibitor of protein kinase C, failed to decrease [³H]QNB binding.5. Stearylamine, an analogue of Sphingosine, was as effective as Sphingosine in inhibiting [³H]QNB binding.6. These results suggest that Sphingosine inhibits muscarinic cholinergic receptor binding by a mechanism that is independent on extracellular Ca²⁺ and protein kinase C.     

β-Amyloid (25-35) or Substance P Stimulates [3H]MK-801. Binding to Rat Cortical Membranes in the Presence of Glutamate and Glycine

Abstract: Micromolar concentrations of β-amyloid (25–35) or substance P stimulated [³H] MK-801 binding in the presence of low concentrations of glutamate (1 γM) and glycine (0.02 γM). Unlike polyamines spermine and spermidine, neither β-amyloid (25–35) nor substance P increased [³H] MK-801 binding in the presence of maximally stimulating concentrations of glutamate and glycine. 5,7-Dichloro-kynurenic acid, CGS-19755, and arcaine completely inhibited the stimulated [³H] MK-801 binding. There was an apparent decreased potency of the [³H] MK-801 binding inhibition curve for 5,7-dichlorokynurenic acid, but not CGS-19755 or arcaine, in the presence of either β-amyloid (25–35) or substance P. The compounds do not appear to act through the strychnine-insensitive glycine binding site because neither β-amyloid (25–35) nor substance P displaced [³H] glycine binding. Full-length β-amyloid (1-40), up to 10 γM, did not stimulate [³H] MK-801 binding. Concentrations >10 γM could not be tested because they formed large aggregate precipitates in the assay. The data indicate that β-amyloid (25–35) or substance P does not stimulate [³H] MK-801 binding at either the N-methyl-D-aspartate, glycine, or polyamine binding sites. Furthermore, the nonpeptide substance P receptor (NK,) antagonist, CP-96,345, did not block β-amyloid (25–35)- or substance P-stimulated [³H] MK-801 binding. Therefore, the effect is not due to an interaction between the substance P receptors and the N-methyl-D-aspartate receptor-operated ionophore. Finally, if these observations can be verified using single-channel recording techniques, they may have implications in the pattern of selective neuronal loss observed in patients with neurodegenerative processes such as Alzheimer's, Parkinson's, and Huntington's diseases.     

Cholinergic muscarinic stimulation of steroidogenesis in bovine adrenal cortex fasciculata cell suspensions

Acetylcholine was found t acutely stimulate cortisol production by bovine fasciculata adrenocortical cell suspensions. This effect was maximal at 10^?4 M acetylcholine concentration, resulted in a 5-fold increase in cortisol production over the control after 1 h incubation, and represented about one fifth of the ACTH maximal stimulation under the same conditions. Acetylcholine-stimulated steroidogenesis was concentration-dependent (10^?8–10^?5 M), propotional to the cell numbe (5 · 10⁵–2 · 10⁶) and reached a plateau after 30 min incubation. Use of various cholinergic specific agonists and antagonists showed that thet steroidogenic action of acetylcholine was a typical muscarinic effect. This character is in agreement with the previously demonstrated presence of muscarinic receptors in bovine adrenocortical tissue. The steroidogenic effect of acetylcholine required the presence of extracellular calcium in the medium and was impaired upon addition of tetracaine and procaine. No change in cyclic AMP nor cyclic GMP levels could be detected in the system under acetylcholine stimulation. Acetylcholine appeared to exhibit a synergistic in combination with ACTH, and exogenous cyclic AMP; these observations suggest a different mechanism of action for acetylcholine and ACTH and point to a possible cholinergic participation in the regulation of adrenocortical differentiated functions in vivo.     

Calium, prostaglandins and the phosphatidylinositol effect in exocrine gland cells

The hypothesis that arachidonic acid metabolism might be involved in Ca-mobilization mechanisms in exocrine gland cells was investigated. Arachidonate (10⁻⁴M) failed to stimulate protein secretion from slices of pancreas, parotid or lacrimal glands and failed to stimulate ⁸⁶Rb efflux from parotid or lacrimal glands. The stimulation of protein secretion (all three glands) or ⁸⁶Rb efflux (parotid and lacrimal glands) by appropriate secretagogues was unaffected by 10⁻⁵M indomethacin. Eicosatetraynoic acid (2×10⁻⁵M) inhibited ⁸⁶Rb efflux due to carbachol but not that due to physalaemin or ionomycin. Nordihydroguaiaretic acid inhibited lacrimal and parotid gland responses only at high (10⁻⁴M) concentration. Collectively, these results argue against an obligatory role for arachidonate metabolites in Ca-mediated responses of these exocrine glands.In the exocrine glands activation by neurotransmitters (or analogs) of receptors that mobilize cellular Ca also stimulates the incorporation of ³²PO₄ into phosphatidylinositol (1–3). Michell (4,5) has suggested that in some manner this alteration in phospholipid metabolism may be functionally responsible for the opening of surface membrane Ca gates which presumably precedes the expression of a number of Ca-mediated responses by the exocrine cell. That this reaction probably preceeds Ca mobilization is deduced primarily from two experimental observations. First, receptor activation of phosphatidylinositol turnover is not prevented by Ca omission (6–8). Second, the effect is not mimicked by the divalent cationophore A-23187, while other effects of receptor activation are mimicked by this compound (7–9).There has also been some speculation as to the manner in which altered phosphatidylinositol metabolism might be involved in the Ca-gating mechanism (10–14). One such hypothesis suggests that receptor activation may lead to phosphatidylinositol breakdown which in turn leads to the release of free arachidonate (13, 14). As free arachidonate is generally believed to be the rate-limiting substrate for prostaglandin synthesis (15), the resulting prostaglandins might act to mobilize Ca or might act in concert with Ca (13, 14). There is evidence for this hypothesis for the mouse pancreas, where exogenous arachidonate and prostaglandins can stimulate amylase release (13). The effects of arachidonate, carbachol, caerulein and pancreozmin were all antagonized by sub-micromolar concentrations of indomethacin (13), a potent cyclooxygenase inhibitor (15). Additionally, recent reports have demonstrated stimulation by acetylcholine of prostaglandin E synthesis in mouse pancreas (16, 17).The purpose of this study was to examine the general applicability of this hypothesis by investigating the effects of arachidonate and substances that inhibit prostaglandin formation in two other exocrine tissues that show a prominent phosphatidylinositol turnover — the rat parotid and lacrimal glands.     

The NK-1 Receptor and a Calcium-Phospholipid Pathway: Inositol Trisphosphate Production and Calcium Movements Induced by Selective Agonists of Neurokinin Receptors in Rat Parotid Glands

In the rat parotid gland, substance P has been shown to induce a phosphatidylinositol bisphosphate breakdown resulting in an inositol trisphosphate production. These data suggested that substance P activated a phospholipase C and thus mediated its effects through the calcium-phospholipid pathway. To determine which neurokinin (NK) receptor was involved in the substance P response, we have used selective agonists of the different NK receptors and examined their effects on both inositol trisphosphate production and calcium movements. A selective NK-1 receptor agonist, [Sar9Met(O2)11]-substance P, evoked an [3H]inositol trisphosphate production and a rapid and transient 45Ca2+ efflux. On the other hand, selective NK-2 and NK-3 receptor agonists, [beta-Ala8]-NKA(4-10) and [MePhe7]-NKB, respectively, were without effect. We conclude that, in the rat parotid glands, only the NK-1 receptors are coupled to the calcium-phospholipid pathway. The C-terminal part of substance P appeared to be sufficient to stimulate this route because the C-terminal octapeptide, substance P(4-11), mimicked substance P effects on both inositol trisphosphate production and calcium movements. The NK-2 and NK-3 receptors, if present in the rat parotid glands, are not associated with the calcium-phospholipid pathway.     

The relationship of phosphatidylinositol turnover to receptors and calcium-ion channels in rat parotid acinar cells.

To help elucidate the possible role of phosphatidylinositol in the regulation of membrane permeability to Ca2+, the relationship in the rat parotid gland of phosphatidylinositol turnover to hormone receptor binding and to the hormone-mediated increase in K+ permeability (a Ca2+-dependent phenomenon) was investigated. The concentrations of adrenaline and substance P required to stimulate phosphatidylinositol turnover were found to be similar to those required for the Ca2+-mediated change in K+ permeability and for ligand binding. However, in the case of muscarinic (cholinergic) receptor stimulation, the phosphatidylinositol response was better correlated to the increase in membrane permeability to Ca2+, as determined by the change in K+ permeability, than to receptor occupation. Consistent with this relationship between the phosphatidylinositol response and Ca2+-channel activation were results obtained by simultaneous administration of maximal or submaximal concentrations of muscarinic and alpha-adrenergic agonists. The extent of 32P incorporation when stimulated by maximal concentrations of two agonists did not summate, but, rather, was intermediate between the response of either agonist alone. One interpretation for these observations is that the phosphatidylinositol response may not be related to receptor occupation or activation, but may be involved in the Ca2+-gating mechanism itself.