Intracellular Transduction Mechanisms Research Articles

Prostaglandin E2EP2 and EP4 receptor activation mediates cAMP-dependent hyperpolarization and exocytosis of renin in juxtaglomerular cells

PGE(2) and PGI(2) stimulate renin secretion and cAMP accumulation in juxtaglomerular granular (JG) cells. We addressed, at the single-cell level, the receptor subtypes and intracellular transduction mechanisms involved. Patch clamp was used to determine cell capacitance (C(m)), current, and membrane voltage in response to PGE(2), EP2 and EP4 receptor agonists, and an IP receptor agonist. PGE(2) (0.1 micromol/l) increased C(m) significantly, and the increase was abolished by intracellular application of the protein kinase A antagonist Rp-8-CPT-cAMPS. EP2-selective ligands butaprost (1 micromol/l), AE1-259-01 (1 nmol/l), EP4-selective agonist AE1-329 (1 nmol/l), and IP agonist iloprost (1 micromol/l) significantly increased C(m) mediated by PKA. The EP4 antagonist AE3-208 (10 nmol/l) blocked the effect of EP4 agonist but did not alter the response to PGE(2). Application of both EP4 antagonist and EP2-antagonist AH-6809 abolished the effects of PGE(2) on C(m) and current. EP2 and EP4 ligands stimulated cAMP formation in JG cells. PGE(2) rapidly stimulated renin secretion from superfused JG cells and diminished the membrane-adjacent granule pool as determined by confocal microscopy. The membrane potential hyperpolarized significantly after PGE(2), butaprost, AE1-329 and AE1-259 and outward current was augmented in a PKA-dependent fashion. PGE(2)-stimulated outward current, but not C(m) change, was abolished by the BK(Ca) channel inhibitor iberiotoxin (300 nmol/l). EP2 and EP4 mRNA was detected in sampled JG cells, and the preglomerular and glomerular vasculature was immunopositive for EP4. Thus IP, EP2, and EP4 receptors are associated with JG cells, and their activation leads to rapid PKA-mediated exocytotic fusion and release of renin granules.

The brain decade in debate: V-Neurobiology of depression

This paper is a transcription of a virtual symposium on the neurobiology of depression, which was organized by the Brazilian Society for Neuroscience and Behavior (SBNeC). There is a consensus that the monoaminergic theory is still valid, but its initial formulation suffered several modifications to explain more recent findings. Moreover, it is naive to suppose that depression has only one substratum and probably various neurotransmitter systems should be involved. Although nowadays the focus is mainly put on the serotonin (5-HT) system, there are several evidences suggesting an important role for other systems. It is expected that neuroimaging research with more specific tracers will give important information on this subject. The hormonal modulation is another important aspect of this picture, particularly in relation to the gender differences observed in depression. The neurobiological relationship between mania and depression states is also discussed. The intracellular transduction mechanisms are a growing field in depression research, representing the central focus of the “molecular theory” of depression, and it is indicated as the most fruitful theory to the development of really new drugs to treat depression.

Functional Specificity of Ventral Striatal Compartments in Appetitive Behaviors

The nucleus accumbens and its associated circuitry subserve behaviors linked to natural or biological rewards, such as feeding, drinking, sex, exploration, and appetitive learning. We have investigated the functional role of neurotransmitter and intracellular transduction mechanisms in behaviors subserved by the core and shell subsystems within the accumbens. Local infusion of the selective NMDA antagonist, AP-5, into the accumbens core, but not the shell, completely blocked acquisition of a bar-press response for food in hungry rats. This effect was apparent only when infused during the early stages of learning. We have also recently shown that infusion of certain protein kinase inhibitors into the core also impairs learning in the same paradigm. These results suggest that plasticity-related mechanisms within the accumbens core, involving glutamate-linked intracellular second messengers, are important for response-reinforcement learning. In contrast to the core, which primarily connects to somatic motor output systems, the shell is more intimately linked to viscero-endocrine effector systems. We have shown that both AMPA and GABA receptors within the medial shell (but not the core) are critically involved in controlling the brain's feeding pathways, via activation of the lateral hypothalamus (LH). This effect is blocked by local inhibition of the LH in double-cannulae experiments and also strongly and selectively activates Fos expression in the LH. These results provide a newly emerging picture of the differentiated functions of this forebrain region and suggest an integrated role in the elaboration of adaptive motor actions.

Abnormal transduction mechanisms in pituitary adenomas.

Pituitary adenomas are differentiated tumors expressing their appropriate mature hormone. Tumoral cells sometimes present with a defective physiological inhibitory or stimulatory control, resulting in paradoxical responses or nonresponsiveness to regulatory neurohormones. These abnormalities can be explained by defects at the intracellular transduction mechanism level. Knowledge of these defective pathways has made progress in the understanding of the pathogenesis of pituitary adenomas possible. The discovery of mutations of Gs alpha named gsp oncogenes in 40% of human somatotropinomas represents one of the most important advances in this field. Other molecular alterations were identified but are rare and sporadic and the pathogenesis of pituitary adenomas remains largely unknown. Abnormal transduction mechanisms may also result in a variable sensitivity of tumors to pharmacological therapy. The dopamine agonist, bromocriptine, is able to normalize blood PRL levels and to reduce tumor size in the majority of patients with prolactinoma, but is ineffective in 8-15% of them. Under physiological conditions, PRL secretion is under the tonic inhibitory control of dopamine which binds D2 receptors negatively coupled to adenylyl cyclase. Several defects in the dopaminergic transduction pathways participate in this bromocriptine resistance. The mean D2-binding site density is decreased to 50% as compared to responsive tumors. This loss of D2 receptors can account for a lower transcription level of its gene and is accompanied by modifications in the messenger alternative splicing; the D2 short isoform receptor expression decreases preferentially. A reduction in Gi2 alpha protein expression is also observed and is correlated to that of the D2 receptor. Finally, the pituitary-specific transcription factor Pit-1 expression is affected. A highly significant correlation was seen between the D2 receptor mRNA and Pit-1 mRNA levels. These defects observed on many levels of the dopaminergic transduction cascade may be the first steps in the loss of the functional features of differentiated tumors toward more proliferative tumors.

Regulation of serotonin-facilitated dopamine release in vivo: the role of protein kinase A activating transduction mechanisms.

Recent neuroanatomical, biochemical, and electrophysiological studies suggest that serotonin (5HT) can modulate dopaminergic function at the level of the cell body and the nerve terminal. The receptor subtypes, regulatory processes, and intracellular transduction mechanisms mediating these interactions remain to be characterized. The potential involvement of cAMP in mediating 5HT-facilitated increases in extracellular levels of striatal dopamine (DA) was assessed using in vivo microdialysis. Local infusion of 0.4 nmol 5HT delivered via probes located in the anterior striata of chloral hydrate-anesthetized male rats significantly increased extracellular DA levels to approximately 700% of basal control levels. Local, intrastriatal infusion of either 2 nmol forskolin, 2 nmol rolipram, 100 nmol isobutylmethylxanthine, or 200 nmol dibutyryl cAMP significantly increased basal DA levels to 28 +/- 3%, 143 +/- 5%, 56 +/- 7%, and 52 +/- 3% above control levels, respectively. Additionally, coperfusion of any of these agents with 5HT significantly decreased the 5HT-facilitory effect on DA release to approximately 50% of observed 5HT controls. The current results suggest a role for the cAMP second-messenger systems in modulating 5HT-facilitated DA release.

Serotonin (5-HT) and pain: A reappraisal of its role in the light of receptor multiplicity

Centrifugal serotonergic pathways running from the rostroventral medulla (RVM) to the dorsal horn of the spinal cord have long been considered an integral component of mechanisms of ‘descending inhibition’ limiting the access of nociceptive information to higher centres. However, the hypothesis of a generalized antinociceptive role of these serotonergic projections no longer seems tenable. Indeed, multiple serotonin (5-HT) receptor types in the dorsal horn appear to fulfil differential roles in the control of nociception, reflecting their contrasting patterns of coupling to intracellular transduction mechanisms. Further, the importance of actions of 5-HT outside the dorsal horn should not be neglected. These aspects have been synthesized into a global view of the role of 5-HT in the control of nociception.

Paf antagonists block induction of nitric oxide synthase in cultured macrophages and vascular smooth muscle cells.

1. Nitric oxide (NO) synthase inhibitors and Paf antagonists abrogate hypotension in septic shock. The latter may act by blocking intracellular transduction mechanisms in vascular smooth muscle cells and inflammatory cells. We examined the effect of Paf antagonists on expression of inducible NO synthase. 2. A murine macrophage cell line (J774.2) and rat vascular smooth muscle cells (VSMC) were stimulated with lipopolysaccharide (LPS), either alone or in combination with Paf or Paf antagonists, BN 50739 or E-6123. 3. NO synthase activity in J774.2 was measured by the conversion of [3H]L-arginine to [3H]L-citrulline. Nitrite accumulation was measured in the culture medium of J774.2 and VSM. 4. BN 50739 (10 mumol/L and E-6123 (1 mumol#L) both reduced the expression of calcium-independent NO synthase activity and nitrite accumulation, while Paf alone had no effect. 5. Inhibition of NO synthase induction by Paf antagonists might afford therapeutic benefits in the management of septic shock and possibly other cardiovascular disorders.

The pharmacological profile of cloned and stably expressed α1B-adrenoceptor in CHO cells

Using Chinese hamster ovary (CHO) cells stably expressing the α 1B-adrenoceptor (CHO α 1B cells) as a model, we investigated whether the transfected cells that express α 1B subtype of adrenoceptor can show the pharmacologic characteristics as previously defined in native tissues. Radioligand binding studies with 2-[β-(4-hydroxy-3-[ 125I]iodophenyl)ethylamino-methyl]tetralone ([ 125I]HEAT) in CHO α 1B cells showed the similar K i values of the α 1-adrenoceptor selective drugs as previously observed in rat liver and spleen, and that pretreatment with chlorethylclonidine markedly inactivated the binding sites (94.7–98.6%). In CHO α 1B cells α 1-adrenoceptor agonists caused a dose-dependent increase in transients of cytosolic Ca 2+ concentrations ([Ca 2+] i), and the potency order of antagonists in inhibiting norepinephrine-induced [Ca 2+] i response was similar to that observed in radioligand binding assays. In summary, the present study shows that the ligand binding property, the pharmacological characteristics and the intracellular transduction mechanisms of α 1B-adrenoceptors stably expressed in CHO cells appear to be the same as those defined in native tissues. Thus they can be a useful model system for further characterization of the receptor as well as for the development of specific ligands.

Verapamil decreases lymphocyte protein kinase C activity in humans

To determine if clinically used doses of the calcium antagonist verapamil measurably alter intracellular transduction mechanisms associated with the phosphatidylinositol pathway, lymphocyte protein kinase C activity was determined in subjects in a drug-free state, after 1 week of verapamil treatment (120 mg three times daily) and after a second week of verapamil treatment (240 mg sustained-release preparation once daily). Nine healthy male volunteers were studied and in these subjects baseline protein kinase C activity (mean +/- SEM; 5.07 +/- 0.76 pmol/microgram protein/min) tended to decrease after 1 week (3.50 +/- 0.20 pmol/micrograms protein/min) and was significantly decreased after 2 weeks (3.14 +/- 0.27 pmol/micrograms protein/min; p < 0.05 from baseline) of verapamil treatment. These data indicate that verapamil, at usual clinical doses, decreases protein kinase C activity in a marker tissue, the circulating lymphocyte. If protein kinase C activity in this tissue is a surrogate for other verapamil target tissues, such as vascular smooth muscle and heart muscle, these findings may provide insight into the in vivo mechanism by which verapamil decreases protein synthesis, limits cell growth, and reverses cellular hypertrophy in these tissues.

Biologically active domain of the secreted form of the amyloid beta/A4 protein precursor.

The amyloid beta/A4 protein precursor (APP), a large transmembrane protein, is expressed ubiquitously in many organisms, as well as in a variety of cultured cells. Studies of the synthesis and processing of APP have revealed several intricate metabolic pathways for this protein. One of these pathways involves the cleavage of APP in the middle of the beta/A4 domain and results in the secretion of the large amino-terminal portion of the protein. The biological function of this secreted form of APP has been the subject of intense investigation by several groups and various activities have been described for the different domains of APP studied. Our initial approach was to create a fibroblast cell line in which APP expression is dramatically reduced. These fibroblasts, called A-1, have a very slow growth rate. Addition of exogenous APP in the medium of A-1 cells restores their growth to the level of normal parent fibroblasts, demonstrating a growth factor-like activity for the secreted form of APP. Using APP fragments made in bacteria as well as synthetic peptides, we have been able to locate the active site of APP within a domain of 17 amino-acids (Ala319-Met335). This domain of APP can stimulate neurite extension of cultured neuroblastoma cells and it is proposed that APP mediates this effect through binding to a cell surface receptor, triggering intracellular transduction mechanisms. Thus, the secreted form of APP can function as a growth and/or differentiation factor and the site involved in these activities is within a 17-mer domain in the middle of the molecule. Our current lines of research seek to further characterize the mechanisms of APP function as well as its activity in vivo.

Defective chemotaxis and calcium response in localized juvenile periodontitis neutrophils.

Localized juvenile periodontitis (ljp) is an early onset form of periodontal disease characterized by unique localization to first molars and incisors and a high prevalence of neutrophil abnormalities, particularly chemotaxis. The intracellular transduction mechanisms that follow receptor-ligand coupling on the neutrophil surface and lead to chemotaxis are not clearly established. Chemotaxis and phagocytosis are modulated by a variety of receptors and involve several activation pathways; the role of intracellular calcium as a presumptive second messenger and mediator of these events is well established. The putative effector mechanisms for the chemotactic receptor of neutrophils also include the possible activation of a phospholipase, protein kinase C, methyltransferase, or adenylate cyclase. In normal neutrophils, a phosphoinositide pathway initiated by phospholipase C, which results in the activation of protein kinase C via diacylglycerol and the generation of IP3, has been implicated. In order to better understand the stages of neutrophil transduction, fluorescent probes were used to monitor neutrophil calcium changes. Chlorotetracycline (CTC) was used as an indirect probe of intracellular membrane-bound pool of calcium stores, and Quin-2 was used to monitor cytosolic free calcium levels of FMLP stimulated normal and LJP neutrophils. The results indicate that the early phase of the calcium response affiliated with the release of intracellularly sequestered calcium appears intact in LJP neutrophils, as the CTC fluorescence changes were similar to control values. The second phase of the calcium response, associated with membrane channel activation and an influx of extracellular calcium, appeared compromised in the neutrophils of the LJP population.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of vascular permeability factor/vascular endothelial growth factor receptors on mononuclear phagocytes

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4 degrees C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (approximately 20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were paralleled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37 degrees C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125I- VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-labeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.

Characterization of Vascular Permeability Factor/Vascular Endothelial Growth Factor Receptors on Mononuclear Phagocytes

AbstractVascular permeability factor/vascular endothelial growth factor (VPF/VEGF) is a polypeptide mediator, elaborated by certain tumors and other cell types, that exerts multiple effects on endothelium via interaction with a class of high-affinity binding sites. In this report, the interaction of VPF/VEGF with human mononuclear phagocytes (MPs) is characterized. Radioligand binding studies at 4°C showed the presence of a single class of binding sites, kd approximately 300 to 500 pmol/L (~20 times lower affinity than the high-affinity binding site on endothelial cells [ECs]), the occupancy of which correlated with VPF/VEGF-induced MP migration and expression of tissue factor. These binding results were parallelled by functional experiments which indicated that the same VPF/VEGF preparations were about an order of magnitude less effective in stimulating MP chemotaxis than in inducing EC proliferation. When MPs with surface-bound 125I-VPF/VEGF were warmed to 37°C, endocytosis and degradation occurred. Occupancy of VPF/VEGF binding site resulted in subsequent activation of intracellular signal transduction mechanisms, as shown by an increase in MP intracellular calcium concentration. Cross-linking studies with 125I-VPF/VEGF showed a new high-molecular weight band (corresponding to putative 125l-VPF/VEGF-receptor complex), the appearance of which was blocked by excess unlabeled VPF/VEGF. Consistent with these results, immunoprecipitation of 32PO4-Iabeled MPs exposed to VPF/VEGF showed a single band of similar mobility, not seen in untreated controls. These results demonstrate that the interaction of VPF/VEGF with MPs, though of lower affinity than that observed with ECs, also results from interaction of the polypeptide with a specific cell-surface protein and leads to activation of intracellular transduction mechanisms.

Testosterone Induces an All-or-None, Exponential Increase in Arginine Vasopressin mRNA in the Bed Nucleus of Stria Terminalis of the Hypogonadal Mouse

The bed nucleus of the stria terminalis (BNST) in rodents contains arginine vasopressin (AVP) neurons which project to the lateral septum (LS) and habenula (LH) and are thought to be important for social recognition or memory. In previous studies we demonstrated that AVP immunoreactivity in the LS and LH is absent in the hypogonadal ( hpg) mouse and that AVP immunoreactivity in the LH and LS of this mutant can be induced by testosterone. The aim of the present study was to determine whether this action of testosterone was due to the induction of AVP gene expression in BNST neurons and if so the time taken for testosterone to exert its effect. We found that exposure to supraphysiological concentrations of testosterone for 6-12 days caused an exponential, 50-fold increase in the number of cells that expressed AVP mRNA in the BNST in hpg mice. The action of testosterone is "all-or-none" in that the level of AVP mRNA in the AVP-positive cells was similar at all times after testosterone implantation and also similar or less than that in the few AVP-positive cells in animals not treated with testosterone. The relatively long time taken for testosterone to exert its effect on AVP mRNA in BNST neurons is comparable to its effects on AVP immunoreactivity in the LH and LS and on behaviour and suggests that the action of testosterone may be mediated by indirect or slow intracellular transduction mechanisms.

Glucocorticoids regulate the development of intracellular signaling: Enhanced forebrain adenylate cyclase catalytic subunit activity after fetal dexamethasone exposure

Although glucocorticoids cause growth retardation and interfere with cell development, selective promotion of some aspects of cell function also has been reported. The current study examines whether glucocorticoids enhance intracellular transduction mechanisms mediated by adenylate cyclase in the developing forebrain, a region in which steroids have been shown to interfere with cell replication, maturation, and growth. Pregnant rats were given dexamethasone at doses spanning the threshold for growth impairment (0.05, 0.2, and 0.8 mg/kg) on gestational days 17, 18, and 19, and development of adenylate cyclase was evaluated in membrane preparations, using four different activity measures: basal adenylate cyclase in the absence or presence of GTP, maximal G-protein activation by fluoride in the presence of GTP, and stimulation mediated by forskolin-Mn 2+, which bypasses the G-proteins. Prenatal exposure to dexamethasone produced a dose-dependent impairment of body growth, with smaller deficits in forebrain weights (brain sparing) indicative of systemic toxicity. Basal adenylate cyclase activity was unaffected by dexamethasone treatment, regardless of whether GTP was present in the assay. Similarly, fluoride stimulation developed normally in all dexamethasone groups. However, forskolin-Mn 2+-stimulated activity was significantly enhanced in a dose-dependent fashion. These results suggest that glucocorticoids serve as positive factors for the development of adenylate cyclase catalytic subunit activity, independently of their adverse effects on general growth and development; thus, these hormones may be a primary regulator of cell signaling during early development.

Changes in ventricular 1,2-diacylglycerol content in rats following monocrotaline treatment.

1,2-Diacylglycerol may initiate cardiac hypertrophy, probably by activating protein kinase C. To test this hypothesis we determined the 1,2-diacylglycerol content of hypertrophied tissue. Rats were treated with monocrotaline and developed severe right ventricular hypertrophy followed by congestive heart failure. 1,2-Diacylglycerol content and fatty acid composition, DNA concentrations, and RNA concentrations in the right ventricle from monocrotaline treated rats were compared with values obtained from the left side or from control rats. During the first week, the right ventricle showed no significant change in 1,2-diacylglycerol content and a small increase in RNA concentration. However, the 1,2-diacylglycerol content was significantly increased by 55% at two weeks after monocrotaline injection, when DNA and RNA synthesis was also enhanced to its highest level when compared with control rats (37% and 18%, respectively). At four weeks after monocrotaline injection, conversely, the 1,2-diacylglycerol content was decreased by 25% in the right ventricle from monocrotaline treated rats, most of which had pleural and peritoneal effusions indicating congestive heart failure, although RNA synthesis was sustained at a high level. The fatty acid composition of 1,2-diacylglycerol did not differ significantly between the right and left ventricles or control rat ventricles. These results suggest that 1,2-diacylglycerol accumulation is associated with development of hypertrophy in monocrotaline treated rats. In contrast, at a stage of congestive heart failure 1,2-diacylglycerol production decreased, suggesting that intracellular transduction mechanisms may be attenuated.

Trans-ACPD induces a slow post-stimulus inward tail current (I ADP) in guinea-pig olfactory cortex neurones in vitro

The metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) produced a slow, persistent excitation of guinea-pig olfactory cortical neurones in vitro, and the appearance of a prominent post-stimulus afterdepolarization. The corresponding slow inward tail current (I ADP) revealed under voltage clamp was insensitive to tetrodotoxin (or atropine) but was blocked by Cd 2+ or tetrabutylammonium. The I ADP properties resembled those of the slow inward tail current induced by muscarinic agonists in these neurones, suggesting a common intracellular transduction mechanism.

Intracellular transduction mechanisms for the slow synaptic events.

Electrical activities of the postganglionic neurons in the superior cervical ganglia of rabbits are modulated in various ways following activation of the subtypes of muscarinic acetylcholine receptors. (i) M1 receptors mediate a slow depolarization consisting of at least three types of ionic conductance changes, and one of these is possibly mediated by cyclic GMP. (ii) M2 receptors mediate a slow hyperpolarization that seems to be generated by inositol triphosphate derived from phosphatidylinositol breakdown. (iii) M2 receptors also cause, through an activation of C kinase, a suppression of Ca entry during action potentials that results in a characteristic change in the action potentials and thereby modulates excitability of superior cervical ganglion neurons. Each subtype of muscarinic receptors thus regulates different pathways of intracellular transduction and modulates the electrical signaling of sympathetic neurons.

Trans-ACPD induces a slow post-stimulus inward tail current (I ADP) in guinea-pig olfactory cortex neurones in vitro

The metabotropic glutamate receptor agonist trans-(±)-1-amino-1,3-cyclopentanedicarboxylic acid (trans-ACPD) produced a slow, persistent excitation of guinea-pig olfactory cortical neurones in vitro, and the appearance of a prominent post-stimulus afterdepolarization. The corresponding slow inward tail current (I ADP) revealed under voltage clamp was insensitive to tetrodotoxin (or atropine) but was blocked by Cd 2+ or tetrabutylammonium. The I ADP properties resembled those of the slow inward tail current induced by muscarinic agonists in these neurones, suggesting a common intracellular transduction mechanism.

Ca2+]i imaging in PC12 cells: multiple response patterns to receptor activation reveal new aspects of transmembrane signaling.

Fura-2 imaging microscopy was used to study [Ca2+]i in nerve growth factor-differentiated PC12 cells exposed to agonists (bradykinin, carbamylcholine, and ATP) binding to receptors coupled to polyphosphoinositide hydrolysis. With all the treatments employed, the response to an individual agonist was often incomplete, i.e., composed of either release from intracellular stores or influx only. In individual cells the responses were closely similar when only one and the same agonist was employed, and markedly heterogeneous, with considerable variation of the release/influx ratio, when different agonists were delivered in sequence. In a recently isolated PC12 cell clone, heterogeneity of the receptor-induced [Ca2+]i responses was markedly lower than in the overall population, although the release/influx ratio was still variable. We conclude that the large response heterogeneity observed in the overall PC12 cell population is due (a) to the coexistence of multiple clones; and (b) to the variable activation of intracellular transduction mechanisms.