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M L Toews

Publications and source records attributed to M L Toews.

At least 19 recordsLinked to original sources
PubMed1996

Regulation of hamster alpha 1B-adrenoceptors expressed in Chinese hamster ovary cells.

Chinese hamster ovary (CHO) cells were stably transfected to express the hamster alpha 1B-adrenoeceptor, and the function and agonist-induced regulation of the binding properties of these receptors were characterized. The cells expressed approximately 230,000 receptors per cell, with a KD for [3H]prazosin of 140 pM. In assays of competition by epinephrine for [3H]prazosin binding to receptors on intact cells, 88% of the receptors were in a low affinity form. The protein kinase C activator phorbol 12-myristate, 13-acetate (PMA) did not further increase the fraction in the low affinity form, but the protein kinase C inhibitor staurosporine reduced the low affinity fraction to 51%. In sucrose density gradient centrifugation assays of receptor internalization, the percentage of receptors in the light vesicle fraction was 25% for control cells, 53% for epinephrine-pretreated cells, 44% for PMA-pretreated cells, and 53% for cells pretreated with epinephrine plus PMA. Staurosporine completely blocked PMA-induced internalization, but only partially inhibited epinephrine-induced internalization. These results suggest a relationship between low affinity binding and internalization for alpha 1B-adrenoceptors and the involvement of protein kinase C in both processes. Longer-term (24 h) exposure of cells to epinephrine induced an unexpected up-regulation of receptor density of approximately 2-fold that was accompanied by an increase in maximal agonist-stimulated phosphoinositide turnover. These studies document several regulatory differences between alpha 1B-adrenoceptors expressed in transfected CHO cells and those natively expressed in DDT1 MF-2 hamster smooth muscle cells, and they provide additional information on the molecular mechanisms involved in agonist-induced regulation of alpha 1B-adrenoceptors.

Adrenergic alpha-Agonists↗
PubMed1995

Lysophosphatidic acid regulation of cyclic AMP accumulation in cultured human airway smooth muscle cells.

The effects of the simple bioactive lipid mediator lysophosphatidic acid (LPA) on cAMP accumulation were investigated in cultured human airway smooth muscle cells (ASMC). Pretreatment of cells with LPA induced an increase in subsequent stimulation of cAMP accumulation by forskolin and by isoproterenol. When included during the assay of cAMP accumulation rather than as a pretreatment, LPA inhibited forskolin stimulation but enhanced isoproterenol stimulation. Both effects of LPA on forskolin stimulation were completely blocked by pertussis toxin treatment, whereas the effects on isoproterenol stimulation appeared relatively insensitive to pertussis toxin. The protein kinase C activator phorbol-12-myristate-13-acetate (PMA) sensitized forskolin stimulation to a similar extent as did LPA, and the combination of LPA plus PMA caused markedly more sensitization than either agent alone. In contrast, PMA inhibited isoproterenol stimulation and markedly decreased the sensitization induced by LPA. Serum also induced sensitization, and sensitization by LPA plus serum was no greater than that with LPA alone. LPA-induced sensitization appeared to be independent of protein kinase C activation because it was unchanged in cells treated to down-regulate protein kinase C. LPA also stimulated polyphosphoinositide hydrolysis, and this stimulation was partially inhibited by pertussis toxin treatment. These results suggest that LPA activates receptors coupled to both the pertussis toxin-sensitive G protein Gi and the pertussis toxin-insensitive G protein Gq. The complex effects of LPA, PMA, and pertussis toxin on cAMP accumulation in these cells are consistent with the expression of the type 2 isozyme of adenylyl cyclase in these cells.

Cells, Cultured↗
PubMed1994

Inhibition of invasion of murine mammary carcinoma cells by the tyrosine kinase inhibitor genistein.

Tyrosine kinases are ubiquitous enzymes that have been shown to be involved in many cellular functions, including growth and differentiation. Recent studies have shown that they are also involved in integrin signal transduction pathways. Since integrins are known to be involved in cellular adhesion and thus in invasion and metastasis, the possible involvement of tyrosine kinases in invasion was tested. Tumor cell invasion was measured using filter inserts coated with Matrigel, a substance that closely resembles the natural basement membrane. A highly metastatic subline of BALB/c mammary carcinoma (410.4) cells was shown to invade nearly three times as much as a low metastatic subline (168.1). Genistein, an inhibitor of tyrosine kinases, was found to inhibit invasion of 410.4 cells with an EC50 of approximately 1 microM. At a concentration of 37 microM, there was almost complete inhibition of invasion by genistein, whereas the structural analog, daidzein, which does not inhibit tyrosine kinases, had only a small effect. At higher concentrations (370 microM), daidzein also caused marked inhibition. Genistein was able to inhibit invasion at concentrations having little effect on cell growth. However, for daidzein, most of the effect on invasion was apparently due to its effect on growth inhibition. The relatively specific effect of genistein to inhibit tumor invasion suggests a role for tyrosine phosphorylation in this process. Genistein or other tyrosine kinase inhibitors may be effective inhibitors of tumor invasion and metastasis.

Animals↗
PubMed1994

Neuropeptide Y promotes GTP photo-incorporation into a 55 kDa protein.

Incubation of bovine hippocampal membranes with [alpha-32P]GTP and exposure to ultraviolet light resulted in the labelling of seven species with apparent molecular masses of 200, 74, 55, 53, 50, 43 and 40 kDa. Labelling of the 55 kDa species was greatly enhanced in the presence of carboxyl terminal fragments [neuropeptide Y-(18-36)] of neuropeptide Y. Labelling occurred with [alpha-32P]GTP but not [alpha-32P]ATP. A group of putative direct G protein activating peptides including mastoparan, melittin, substance P and adrenocorticotropic hormone (ACTH)-(1-24), were also able to stimulate the labelling of this protein. Labelling of the 55 kDa protein could be demonstrated in bovine brain but not peripheral tissues. Western blot analysis using an antibody against the common alpha subunit of G proteins recognized a protein co-migrating with the 55 kDa GTP-binding protein. These findings demonstrate the existence of a previously uncharacterized neuronal protein, with an apparent molecular mass of 55 kDa, that binds GTP in response to neuropeptide Y and other peptides.

Affinity Labels↗
PubMed1994

TGF-beta 1 modulates beta-adrenergic receptor number and function in cultured human tracheal smooth muscle cells.

Pretreatment of cultured human tracheal smooth muscle cells with transforming growth factor-beta 1 (TGF-beta 1) decreased adenosine 3',5'-cyclic monophosphate (cAMP) accumulation by intact cells stimulated with the beta-adrenergic agonist isoproterenol. The maximal inhibition of isoproterenol-stimulated cAMP accumulation by TGF-beta 1 was 31 +/- 3%, and the mean effective concentration (EC50) of TGF-beta 1 was approximately 1.5 pM. TGF-beta 1 decreased the maximal response to isoproterenol but did not change the EC50 value of isoproterenol. TGF-beta 1 did not change cAMP accumulation stimulated by forskolin. TGF-beta 1 pretreatment decreased isoproterenol-stimulated adenylyl cyclase activity measured in broken cell preparations, but did not change the fluoride-stimulated adenylyl cyclase activity. Together these results suggest that the TGF-beta 1 effect is not by direct inhibition of adenylyl cyclase or by decreased activity of the stimulatory GTP-binding protein. Saturation binding experiments with the beta-adrenergic receptor radioligand [125I]iodopindolol showed that TGF-beta 1 pretreatment decreased the beta-adrenergic receptor number. The protein synthesis inhibitor cycloheximide abolished the effect of TGF-beta 1 on both cAMP accumulation and on beta-adrenergic receptor number, indicating that protein synthesis is involved. These results suggest that TGF-beta 1 in the lung could play a role in changing the responsiveness of airway smooth muscle cells to endogenous catecholamines and to beta-adrenergic agonists used in therapy.

Adenylyl Cyclases↗
PubMed1994

Intact cell binding properties of cells expressing altered beta-adrenergic receptors.

During the course of equilibrium competition binding assays with intact cells, agonists induce conversion of beta-adrenergic receptors (BARs) from a native form with high affinity for agonists to a form with a markedly lower apparent affinity. The roles of receptor internalization, receptor-Gs coupling, and receptor phosphorylation in this agonist-induced conversion to the low affinity form were investigated. Agonist and antagonist competition for [125I]iodopindolol binding to intact cells was measured in mouse L cells expressing wild-type BARs (C+I+), mutated BARs that do not couple to Gs but do internalize (C-I+), and mutated BARs that do not couple to Gs and do not internalize (C-I-). For C+I+ and C-I+ cells, most of the receptors exhibited apparent affinities for the agonist isoproterenol that were 500-900-fold lower in equilibrium assays with intact cells than in short-time assays with intact cells or in equilibrium assays with isolated membranes, similar to previous results with cells expressing native BARs. The extent of conversion to this lower affinity form for C-I- cells was markedly decreased. Binding properties for the antagonist metoprolol were similar for all three BARs in both short-time and equilibrium assays. Isoproterenol competition in short-time and equilibrium assays also was compared in Chinese hamster fibroblasts expressing wild-type BARs, mutated BARs that lack BAR kinase sites, mutated BARs that lack cAMP-dependent protein kinase sites, and mutated BARs that lack both types of phosphorylation sites. All three BAR phosphorylation mutants showed only small but significant decreases, relative to the wild-type BAR, in the extent of conversion to the low affinity form. These results provide additional evidence that receptor internalization is the major determinant for the conversion of intact cell BARs to the low affinity form. Receptor phosphorylation may play a minor role in conversion to the low affinity form, whereas receptor coupling to Gs is apparently not required.

Adrenergic beta-Agonists↗
PubMed1993

Down-regulation of alpha 2-adrenoceptor subtypes.

To characterize further the alpha 2-adrenoceptor subtypes in terms of their regulation, monolayers of cells expressing either the alpha 2A (CHO-A2AR cells) or alpha 2C (OK cells) subtype were preincubated with norepinephrine for various times and the extent of receptor down-regulation was assessed. Exposure to 30 microM norepinephrine caused a similar time course and extent of down-regulation (approximately 50%) in both cells lines. The extent of down-regulation caused by 0.3 microM norepinephrine in OK cells was similar to that with 30 microM norepinephrine in CHO-A2AR cells, although the time course was somewhat slower. Reversal of the down-regulation of the alpha 2-adrenoceptor caused by 30 microM norepinephrine was more rapid in the CHO-A2AR than in the OK cell. With 0.3 microM norepinephrine, reversal of down-regulation of the alpha 2-adrenoceptor in the OK cell was slightly faster than that of the CHO-A2AR cell with 30 microM norepinephrine. These data indicate that although norepinephrine is more potent in causing down-regulation of the alpha 2C (OK cells) as compared to the alpha 2A subtype (CHO-A2AR cells), the time courses for down-regulation and its reversal are similar for the two subtypes.

Animals↗
PubMed1993

Agonist-induced desensitization of beta-adrenoceptors of bovine bronchial epithelial cells.

1. Cultured bovine bronchial epithelial cells express a large number of beta-adrenoceptors of the beta 2 subtype which are coupled to activation of adenylate cyclase and synthesis of cyclic AMP. The mechanisms involved in agonist-induced desensitization of beta-adrenoceptor function in these cells were investigated. 2. Preincubation of cells with the beta-adrenoceptor agonist isoprenaline induced a rapid desensitization of adenylate cyclase activity stimulated by isoprenaline (40% at 30 min) with little or no decrease in forskolin stimulation or in the number of beta-adrenoceptors detected by radioligand binding, indicating that uncoupling of beta-adrenoceptors from adenylate cyclase had occurred. 3. Uncoupling of beta-adrenoceptors from the stimulatory guanine-nucleotide-binding protein Gs was indicated by the loss of GTP effects on agonist competition for radioligand binding. Results from subcellular fractionation by sucrose density gradient centrifugation suggest that rapid internalization or sequestration of beta-adrenoceptors occurred along with the desensitization. 4. Preincubation with isoprenaline for longer times led to a marked decrease in total receptor number (85% at 18 h), indicating that receptor down-regulation also occurs. Recovery of receptors after down-regulation was prevented by cycloheximide and by actinomycin D, suggesting that degradation of both receptor protein and receptor mRNA may be involved in down-regulation.

Adenylyl Cyclases↗
PubMed1993

Lysophosphatidic acid mimics serum-induced sensitization of cyclic AMP accumulation.

Pretreatment of 1321N1 human astrocytoma cells with serum induces a pronounced increase in subsequent stimulation by forskolin and other agents of intracellular cyclic AMP accumulation, a phenomenon referred to as sensitization (Mol. Pharmacol. 39, 399-406, 1991). Pretreatment of these cells with lysophosphatidic acid induced sensitization to a similar extent as that with serum (approximately fivefold for forskolin stimulation and twofold for isoproterenol and prostaglandin E1 stimulation), with half-maximal effects at approximately 30 nM lysophosphatidic acid. Phosphatidic acid was effective but less potent whereas other lipids were ineffective. Sensitization by serum and by lysophosphatidic acid were almost completely inhibited by pertussis toxin pretreatment and partially inhibited by prolonged phorbol ester exposure to induce protein kinase C down-regulation. Among nine cell lines tested, those that exhibited sensitization with serum showed comparable sensitization with lysophosphatidic acid. The effects of both lysophosphatidic acid and serum were markedly inhibited by treatment with phospholipase B but only minimally altered with phospholipases A2, D, and C. Exposure of cells to phospholipase C alone induced approximately threefold sensitization, but both serum and lysophosphatidic acid were able to induce further three- to fourfold sensitization above that induced by phospholipase C alone. In contrast, the effects of serum and lysophosphatidic acid were not additive with each other. Together these results suggest that lysophosphatidic acid or a closely related compound present in serum is the factor responsible for sensitization of the cyclic AMP pathway.

Alprostadil↗
PubMed1993

Radioligand binding methods: practical guide and tips.

Radioligand binding assays are a relatively simple but extremely powerful tool for studying receptors. They allow an analysis of the interactions of hormones, neurotransmitters, growth factors, and related drugs with the receptors, studies of receptor interactions with second messenger systems, and characterization of regulatory changes in receptor number, subcellular distribution, and physiological function. As a result, these assays are widely used (and often misused) by investigators in a variety of disciplines, including pharmacology, physiology, biochemistry, immunology, and cell biology. This article presents a broad overview of the radioligand binding assay technique, primarily for the investigator who has limited experience with this technique. Practical guidelines for setting up a new assay are presented, including the receptor preparation to be used, choice of appropriate radioligand, optimizing assay conditions, and appropriate methods for data analysis. Tips for avoiding some of the common pitfalls in application of these assays are also included. The primary focus is on radioligand binding assays of membrane-bound receptors studied in membrane preparations. However, similar assay techniques can be used to study receptors on intact cells. The unique advantages and disadvantages of these intact cell binding assays are also discussed. In particular, the occurrence of regulatory changes in receptors during the course of intact cell binding assays is considered, with approaches for circumventing these complications and for using intact cell assays to advantage in studying these regulatory changes.

Animals↗
PubMed1993

Bicarbonate induces sensitization of cyclic AMP accumulation by intact 1321N1 human astrocytoma cells.

Preincubation of 1321N1 human astrocytoma cells in medium buffered with bicarbonate induced 50 to 100% increases in subsequent stimulation of cyclic AMP accumulation by the beta adrenergic receptor agonist isoproterenol and by the direct adenylyl cyclase activator forskolin, compared to cells incubated in buffered medium without bicarbonate. This "bicarbonate-induced sensitization" of cyclic AMP accumulation occurred rapidly, was rapidly reversible and was bicarbonate concentration-dependent. Although protein kinase C activation also induces sensitization in these cells, sensitization by bicarbonate does not appear to involve protein kinase C, because neither protein kinase C down-regulation nor the kinase inhibitor staurosporine prevented sensitization. Pertussis toxin neither mimicked nor prevented the sensitization by bicarbonate, suggesting that pertussis toxin-sensitive guanine nucleotide-binding proteins are not involved. The effect of bicarbonate appears to be on synthesis of cyclic AMP rather than degradation, inasmuch as the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine did not prevent expression of sensitization and because cyclic AMP degradation rates were not altered after bicarbonate incubation. However, increased adenylyl cyclase activity was not retained in broken cell preparations from cells incubated with bicarbonate. These results suggest the occurrence of a novel mechanism through which bicarbonate exposure can regulate adenylyl cyclase activity in intact cells.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Effects of antimycin A on the binding properties of beta and alpha-1 adrenergic receptors measured on intact cells.

Depletion of intracellular ATP with antimycin A has been shown to inhibit the internalization of several types of receptors, including beta adrenergic and muscarinic acetylcholine receptors. The effects of antimycin A treatment on conversion of beta and alpha-1 adrenergic receptors to a form exhibiting low apparent affinity for agonists in assays with intact cells were investigated, because conversion to the low-affinity form has been postulated to result from receptor internalization. Treatment of DDT1 MF-2 hamster smooth muscle cells with antimycin A greatly decreased conversion of beta adrenergic receptors to the form exhibiting low affinity for agonists. Binding of antagonists to intact cell beta adrenergic receptors was not altered by antimycin A. In contrast to the results with beta adrenergic receptors, conversion of alpha-1 adrenergic receptors to the low-affinity form was not inhibited by antimycin A. These results are consistent with the possible involvement of receptor internalization in conversion to the low affinity form for beta adrenergic receptors. They also provide further evidence that different mechanisms may be involved in conversion to the low-affinity form or in the internalization pathway in the case of alpha-1 adrenergic receptors.

Adenosine Triphosphate↗
PubMed1992

Neuropeptide Y inhibits forskolin-stimulated adenylate cyclase in bovine adrenal chromaffin cells via a pertussis toxin-sensitive process.

The effect of neuropeptide Y (NPY) on adenylate cyclase activity and the role of G-proteins mediating NPY's effect were investigated in cultured bovine adrenal chromaffin cells. The equilibrium binding of [125I]NPY to sucrose gradient purified bovine adrenal medulla plasma membranes revealed high- (GTP gamma S sensitive) and low-affinity binding sites with calculated IC50 values of 0.27 nM and 0.14 microM, respectively. Inhibition of forskolin-stimulated cyclic AMP accumulation was dependent upon the NPY concentration (IC50 = 0.9 nM) and independent of cyclic AMP (cAMP) phosphodiesterase activity. NPY-related peptides, except peptide YY, and NPY fragments exhibited minimal inhibitory activity. The inhibitory effect of NPY on forskolin-stimulated adenylate cyclase activity was completely abolished by pretreatment of the cells with pertussis toxin (PTX). Incubation of membranes with PTX and [32P]nicotinamide adenine dinucleotide revealed a protein band with an apparent molecular mass of 41 kDa. The time course and dose dependence of PTX pretreatment for in vitro ADP-ribosylation were similar to those for PTX to attenuate the NPY effect on forskolin-stimulated adenylate cyclase activity. The direct relation between the NPY receptor and the PTX-sensitive G-protein was further shown by the ability of NPY to inhibit PTX-catalyzed in vitro ADP-ribosylation. ADP-ribosylation of the 41-kDa protein was partially inhibited by 5'-guanylylimidodiphosphate and further inhibited by high concentrations of NPY. An antibody against Gi1/i2 alpha 1 recognized two species of which a 41-kDa protein comigrated with the PTX substrate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate Ribose↗
PubMed1992

Serum-induced sensitization of cyclic AMP accumulation in C62B rat glioma cells.

Exposure of C62B rat glioma cells to fresh medium containing fetal bovine serum induced a sensitization of the subsequent ability of isoproterenol and forskolin to stimulate cyclic AMP accumulation, compared to cells exposed to fresh medium without serum. Isoproterenol stimulation was typically increased by 2- to 4-fold and forskolin stimulation by 3- to 5-fold. Sensitization occurred rapidly, was rapidly reversible and appeared to result from an increase in maximal stimulation. A commercial preparation of albumin, purified chromatographically so as to retain bound lipids and other factors, was able to mimic the effect of serum. In contrast to the effects of serum, exposure of cells to phorbol 12-myristate, 13-acetate induced little or no change in forskolin stimulation but a marked desensitization of isoproterenol stimulation that was due primarily to a decrease in potency. Neither the protein kinase C inhibitor staurosporine or overnight exposure to phorbol 12-myristate, 13-acetate to down-regulate protein kinase C prevented serum-induced sensitization. Pertussis toxin almost completely blocked serum-induced sensitization, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in mediating the effects of serum. Sensitization was poorly retained in membrane adenylate cyclase assays. Studies with the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine, direct assays of cyclic AMP degradation by intact cells and assays of phosphodiesterase activity in cell lysates all indicated that degradation of cyclic AMP was decreased in serum-pretreated cells. Thus, both increased cyclic AMP synthesis and decreased cyclic AMP degradation may contribute to sensitization in these cells.

1-Methyl-3-isobutylxanthine↗
PubMed1991

Serum-induced sensitization of cyclic AMP accumulation in 1321N1 human astrocytoma cells.

Exposure of 1321N1 human astrocytoma cells to fresh medium containing fetal bovine serum induced a marked increase in the subsequent ability of isoproterenol and forskolin to stimulate cAMP accumulation in intact cells, compared with cells exposed to fresh medium without serum. This "sensitization" of cAMP accumulation by serum was dose dependent, occurred rapidly, was maintained in the continuing presence of serum, and reversed rapidly upon removal of serum. Preliminary characterization of the sensitizing factor(s) in serum has been performed, but the factor(s) remain to be identified. Sensitization appeared to result from an increase in maximal response and not from changes in the potency of isoproterenol or forskolin. The protein kinase C inhibitor staurosporine inhibited serum-induced sensitization. Furthermore, down-regulation of protein kinase C almost completely eliminated the subsequent ability of serum to induce sensitization, indicating involvement of protein kinase C in the serum effect. Pretreatment of cells with pertussis toxin also markedly reduced subsequent sensitization induced by serum, suggesting involvement of a pertussis toxin-sensitive guanine nucleotide-binding protein in the pathway for serum-induced sensitization. The rate of cAMP degradation was not changed in sensitized cells, but some increase in adenylyl cyclase activity was retained in broken cell preparations from sensitized cells, suggesting increased synthesis of cAMP by adenylyl cyclase as the mechanism for sensitization.

1-Methyl-3-isobutylxanthine↗
PubMed1990

Regulation of histamine H1 receptor-mediated phosphoinositide hydrolysis by histamine and phorbol esters in DDT1 MF-2 cells.

The regulation of histamine-stimulated phosphoinositide turnover by histamine and phorbol esters was examined in intact DDT1 MF-2 cells grown in suspension culture. Histamine increased the incorporation of 32P into phosphatidylinositol (PI) in these cells, and this stimulation was inhibited by the H1 antagonist diphenhydramine but not by the H2 antagonist cimetidine. Pretreatment of cells with histamine or with phorbol 12-myristate 13-acetate (PMA) or other activators of protein kinase C induced a marked decrease in the subsequent stimulation by histamine. PMA, but not histamine, also decreased the ability of epinephrine to stimulate PI labelling through alpha 1-adrenoceptors. Thus, histamine appears to induce homologous desensitization of histamine H1 receptor-mediated PI turnover, whereas direct activation of protein kinase C in the absence of receptor occupancy by agonist induces nonspecific heterologous desensitization of both histamine H1- and alpha 1-adrenoceptor-mediated responses.

Animals↗
PubMed1990

Protein kinase C activators sensitize cyclic AMP accumulation by intact 1321N1 human astrocytoma cells.

Pretreatment of 1321N1 human astrocytoma cells with phorbol 12-myristate-13-acetate or other activators of protein kinase C led to 2.5- to 5-fold increases (sensitization) in subsequent stimulation by forskolin of intracellular cyclic AMP accumulation. These compounds caused much smaller or no increases in receptor-mediated stimulation of cyclic AMP accumulation induced by isoproterenol and by prostaglandin E1. Carbachol and histamine, agonists acting at receptors coupled to polyphosphoinositide turnover in these cells, induced less sensitization of subsequent stimulation by forskolin but greater sensitization of stimulation by isoproterenol and by prostaglandin E1. The specificities of various analogs of phorbol 12-myristate-13-acetate, for induction of sensitization of forskolin stimulation were consistent with involvement of protein kinase C. The effects of protein kinase inhibitors and of down-regulation of protein kinase C activity also indicated involvement of protein kinase C in sensitization of forskolin stimulation, although additional mechanisms are likely to be involved in sensitization of isoproterenol stimulation. Neither pertussis toxin pretreatment nor inclusion of isobutylmethylxanthine during assays of cyclic AMP accumulation were able to prevent or mimic these sensitization phenomena, suggesting that the primary site of modification responsible for sensitization is neither the inhibitory guanine nucleotide-binding protein nor cyclic AMP phosphodiesterase. Sensitization was only observed in assays with intact cells. These results, together with those from our previous study describing protein kinase C-mediated desensitization of broken cell adenylate cyclase activity, indicate that activation of protein kinase C leads to multiple changes in the receptor-stimulated adenylate cyclase signal transduction pathway of these cells.

1-Methyl-3-isobutylxanthine↗