M1 and m2 muscarinic receptor subtypes regulate antidepressant-like effects of the rapidly acting antidepressant scopolamine.

Scopolamine produces rapid and significant symptom improvement in patients with depression, and most notably in patients who do not respond to current antidepressant treatments. Scopolamine is a nonselective muscarinic acetylcholine receptor antagonist, and it is not known which one or more of the five receptor subtypes in the muscarinic family are mediating these therapeutic effects. We used the mouse forced-swim test, an antidepressant detecting assay, in wild-type and transgenic mice in which each muscarinic receptor subtype had been genetically deleted to define the relevant receptor subtypes. Only the M1 and M2 knockout (KO) mice had a blunted response to scopolamine in the forced-swim assay. In contrast, the effects of the tricyclic antidepressant imipramine were not significantly altered by gene deletion of any of the five muscarinic receptors. The muscarinic antagonists biperiden, pirenzepine, and VU0255035 (N-[3-oxo-3-[4-(4-pyridinyl)-1-piper azinyl]propyl]-2,1,3-benzothiadiazole-4-sulfonamide) with selectivity for M1 over M2 receptors also demonstrated activity in the forced-swim test, which was attenuated in M1 but not M2 receptor KO mice. An antagonist with selectivity of M2 over M1 receptors (SCH226206 [(2-amino-3-methyl-phenyl)-[4-[4-[[4-(3 chlorophenyl)sulfonylphenyl]methyl]-1-piperidyl]-1-piperidyl]methanone]) was also active in the forced-swim assay, and the effects were deleted in M2 (-/-) mice. Brain exposure and locomotor activity in the KO mice demonstrated that these behavioral effects of scopolamine are pharmacodynamic in nature. These data establish muscarinic M1 and M2 receptors as sufficient to generate behavioral effects consistent with an antidepressant phenotype and therefore as potential targets in the antidepressant effects of scopolamine.

Allosteric modulation of the muscarinic M4 receptor as an approach to treating schizophrenia.

Current antipsychotics provide symptomatic relief for patients suffering from schizophrenia and related psychoses; however, their effectiveness is variable and many patients discontinue treatment due to side effects. Although the etiology of schizophrenia is still unclear, a leading hypothesis implicates an imbalanced dopaminergic system. Muscarinic acetylcholine (ACh) receptors regulate dopamine levels in key areas of the brain involved in psychosis, with the M(4) subtype emerging as a key regulator of dopaminergic hyperactivity. Unfortunately, no selective small molecule tools exist to provide pharmacological validation of this hypothesis. Here, we describe the discovery of a small molecule modulator, LY2033298, that is highly selective for human M(4) receptors by virtue of targeting an allosteric site on this receptor. Pharmacological assays confirmed the selectivity of LY2033298 for the M(4) receptor and revealed the highest degree of positive allosteric enhancement of ACh potency thus far identified. Radioligand binding assays also show this compound to directly potentiate agonist binding while having minimal effects on antagonist binding. Mutational analysis identified a key amino acid (D(432)) in the third extracellular loop of the human M(4) receptor to be critical for selectivity and agonist potentiation by LY2033298. Importantly, LY2033298 was active in animal models predictive of clinical antipsychotic drug efficacy indicating its potential use as a first-in-class, selective, allosteric muscarinic antipsychotic agent.

Differential expression of excitatory amino acid receptor subtypes in cultured cerebellar neurons.

Using neurotoxicity and inositol phosphate release as criteria for receptor expression, we report the differential expression of excitatory amino acid receptor subtypes in cerebellar granule cells grown in serum-free media containing either high (25 mM) or low (5 mM) KCl. NMDA receptors are expressed in neurons grown in high, but not low, KCl. In contrast, ionotropic quisqualate receptors are expressed in neurons grown in low KCl, but not in those grown in high KCl. Addition of NMDA to cultures containing low KCl appears to mimic high KCl conditions: NMDA receptors are expressed, but ionotropic quisqualate receptors are not. Glutamate and kainate are toxic to cells grown in either condition.

Isoform specific differences in phospholipase C beta 1 expression in the prefrontal cortex in schizophrenia and suicide.

Our previous study demonstrated that phospholipase C beta 1 mRNA was down-regulated in Brodmann's area 46 from subjects with schizophrenia. However, phospholipase C beta 1 protein has also been shown to be lower in Brodmann's area 8 and 9 from teenage suicide subjects, creating a potential confound in interpreting the findings in schizophrenia due to the high suicide rate associated with this disorder. To begin to reconcile and consolidate these findings, in this study, we measured mRNA and protein levels of phospholipase C beta 1 variants a and b in Brodmann's area 46 and Brodmann's area 9 from subjects with schizophrenia, many of whom were suicide completers, and determined the diagnostic specificity of observed findings. Consistent with our previous study, levels of phospholipase C beta 1 a and b mRNA, but not protein, were lower in Brodmann's area 46 from subjects with schizophrenia. In Brodmann's area 9, phospholipase C beta 1a protein levels were lower in subjects with schizophrenia, while phospholipase C beta 1b mRNA was higher and protein was lower in those that had died of suicide. Altered protein levels in Brodmann's area 9 appeared to be diagnostically specific, as we did not detect these changes in subjects with bipolar disorder, major depressive disorder or suicide completers with no diagnosis of mental illness. We further assessed the relationship between phospholipase C beta 1 and levels of muscarinic receptors (CHRMs) that signal through this protein, in both human and Chrm knockout mouse central nervous system tissue, and found no strong relationship between the two. Understanding central nervous system differences in downstream effector pathways in schizophrenia may lead to improved treatment strategies and help to identify those at risk of suicide.

Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI.

Evidence is accumulating that calcium homeostasis and calcium-regulated events may be selectively important in generation and maintenance of the malignant phenotype. CAI, a carboxyamido-triazole with a halogenated benzophenone tail, is a novel inhibitor of receptor-operated calcium influx and arachidonic acid release which inhibits malignant proliferation, invasion, and metastasis. The focus of this investigation was structural analysis of CAI and to determine if the inhibition of calcium influx and arachidonic acid release by CAI and its antiproliferative activity were mediated through the same chemical domains. Four families of molecular modifications of the CAI parent were synthesized: (I) modification or substitution of the triazole ring; (II) removal of the substituted benzophenone tail; (III) dehalogenation or partial truncation of the benzophenone moiety; and (IV) removal of the triazole and altered substitutions of the benzophenone tail. Compounds were tested for the inhibition of calcium influx and arachidonic acid release and inhibition of proliferation and colony formation in soft agar using the malignant CHO line transfected with the m5 muscarinic receptor and the A2058 human melanoma cell line. Only CAI and Group I compounds inhibited stimulated calcium influx, arachidonic acid release, and proliferation. Linear regression analysis of the relationship of the 50% inhibitory concentration values for all compounds in inhibition of calcium influx and arachidonate release was statistically significant (r2 = 0.993). Similarly, a linear relationship was demonstrated between inhibition of calcium influx and inhibition of tumor cell proliferation (r2 = 0.971). Groups II-IV had minimal or no signal or growth inhibitory activity. This investigation provides the first evidence for a coordinate link between calcium influx, calcium-mediated arachidonic acid release, and malignant proliferation and metastasis and constitutes the initial analysis of structurally important domains of the CAI molecule.

The endocannabinoid nervous system: unique opportunities for therapeutic intervention.

The active principle in marijuana, Delta(9)-tetrahydrocannabinol (THC), has been shown to have wide therapeutic application for a number of important medical conditions, including pain, anxiety, glaucoma, nausea, emesis, muscle spasms, and wasting diseases. Delta(9)-THC binds to and activates two known cannabinoid receptors found in mammalian tissue, CB1 and CB2. The development of cannabinoid-based therapeutics has focused predominantly on the CB1 receptor, based on its predominant and abundant localization in the CNS. Like most of the known cannabinoid agonists, Delta(9)-THC is lipophilic and relatively nonselective for both receptor subtypes. Clinical studies show that nonselective cannabinoid agonists are relatively safe and provide therapeutic efficacy, but that they also induce psychotropic side effects. Recent studies of the biosynthesis, release, transport, and disposition of anandamide are beginning to provide an understanding of the role of lipid transmitters in the CNS. This review attempts to link current understanding of the basic biology of the endocannabinoid nervous system to novel opportunities for therapeutic intervention. This new knowledge may facilitate the development of cannabinoid receptor-targeted therapeutics with improved safety and efficacy profiles.

The muscarinic agonist xanomeline increases monoamine release and immediate early gene expression in the rat prefrontal cortex.

BACKGROUND: The muscarinic agonist xanomeline has been shown to reduce antipsychotic-like behaviors in patients with Alzheimer's disease. Because atypical antipsychotic agents increase dopamine release in prefrontal cortex and induce immediate early gene expression in prefrontal cortex and nucleus accumbens, the effect of xanomeline was determined on these indices. METHODS: The effect of xanomeline on extracellular levels of monoamines in brain regions was determined using a microdialysis technique, and changes in expression of the immediate early genes c-fos and zif/268 in brain regions were evaluated using in situ hybridization histochemistry. RESULTS: Xanomeline increased extracellular levels of dopamine in prefrontal cortex and nucleus accumbens but not in striatum. Xanomeline increased expression of c-fos and zif/268 in prefrontal cortex and nucleus accumbens. There was no change in immediate early gene expression in striatum. CONCLUSIONS: Xanomeline increased extracellular levels of dopamine, which is similar to the effects of the atypical antipsychotics clozapine and olanzapine. The regional pattern of immediate early gene expression induced by xanomeline resembled that of atypical antipsychotic agents. Based on the antipsychotic-like activity of xanomeline in Alzheimer's patients and the similarity to atypical antipsychotic agents, we suggest that xanomeline may be a novel antipsychotic agent.

Carbachol-induced reverse transformation of Chinese hamster ovary cells transfected with and expressing the m5 muscarinic acetylcholine receptor.

Reverse transformation was induced in Chinese hamster ovary (CHO) cells transfected with and stably expressing the m5 subtype of the muscarinic acetylcholine receptor when stimulated with the muscarinic agonist, carbachol. Atropine, a muscarinic antagonist, blocked the carbachol-stimulated reverse transformation. CHO cells not transfected with the muscarinic receptor did not change with added carbachol. PMA induced reverse transformation without increasing cAMP accumulation in CHO cells. Carbachol, prostaglandin E2, and cholecystokinin increased cAMP accumulation but only carbachol caused reverse transformation. Carbachol-stimulated cAMP accumulation occurred at a higher concentration (EC50 10 microM) than did carbachol-stimulated reverse transformation (EC50 63 nM). Muscarinic m5 acetylcholine receptor transfected into CHO cells can induce reverse transformation which may be independent of cAMP.

The third intracellular domain of the m3 muscarinic receptor determines coupling to calcium influx in transfected Chinese hamster ovary cells.

The m2 and m3 muscarinic acetylcholine receptors were expressed in CHO cells and were shown to couple to the release of calcium from intracellular stores. The m3 receptor, but not the m2 receptor, also coupled to calcium influx. Chimeric m2/m3 receptors were used to determine the structural domain of the m3 receptor linked to the regulation of calcium influx. It was found that the third intracellular loop of m3 receptor plays a fundamental role in regulating Ca2+ influx predicted to occur through Ca2+ channels located in the plasma membrane in CHO cells.

The rat alpha 2-C4 adrenergic receptor gene encodes a novel pharmacological subtype.

A rat gene and brain cDNA (pA2d) encoding the homologue of the human alpha-C4 adrenergic receptor subtype were isolated and characterized. RNA blots indicate that this gene is expressed in brain, heart and kidney but not in lung, liver or pancreas. Yohimbine, WB-4101 and prasozin all exhibited high affinity for this receptor in binding studies. Clonidine was more potent and efficacious than norepinephrine in inhibiting forskolin-stimulated cAMP production in CHO cells expressing pA2d. Together, these data suggest that the alpha 2-C4 gene product defines a previously undescribed pharmacological subtype of alpha 2-adrenergic receptor.

Cloning and characterization of the rat 5-HT5B receptor. Evidence that the 5-HT5B receptor couples to a G protein in mammalian cell membranes.

A gene encoding a novel G protein-coupled 5-hydroxytryptamine (5-HT) receptor, termed 5-HT5B, was cloned. The ligand binding profile of this receptor is distinct from that of other cloned 5-HT receptors. The 5-HT5B receptor couples to a G protein in COS1 cell membranes; however, activation of the 5-HT5B receptor does not appear to alter either cAMP accumulation or phosphoinositide turnover in a variety of fibroblast cell lines. In the rat brain, 5-HT5B gene expression occurs predominantly in the medial habenulae and hippocampal CA1 cells of the adult. Little expression is seen during embryonic development.

Isolation and measurement of the endogenous cannabinoid receptor agonist, anandamide, in brain and peripheral tissues of human and rat.

Anandamide (arachidonylethanolamide) is a novel lipid neurotransmitter first isolated from porcine brain which has been shown to be a functional agonist for the cannabinoid CB1 and CB2 receptors. Anandamide has never been isolated from human brain or peripheral tissues and its role in human physiology has not been examined. Anandamide was measured by LC/MS/MS and was found in human and rat hippocampus (and human parahippocampal cortex), striatum, and cerebellum, brain areas known to express high levels of CB1 cannabinoid receptors. Significant levels of anandamide were also found in the thalamus which expresses low levels of CB1 receptors. Anandamide was also found in human and rat spleen which expresses high levels of the CB2 cannabinoid receptor. Small amounts of anandamide were also detected in human heart and rat skin. Only trace quantities were detected in pooled human serum, plasma, and CSF. The distribution of anandamide in human brain and spleen supports its potential role as an endogenous agonist in central and peripheral tissues. The low levels found in serum, plasma, and CSF suggest that it is metabolized in tissues where it is synthesized, and that its action is probably not hormonal in nature.

1-(1,2,5-Thiadiazol-4-yl)-4-azatricyclo[2.2.1.0(2,6)]heptanes as new potent muscarinic M1 agonists: structure-activity relationship for 3-aryl-2-propyn-1-yloxy and 3-aryl-2-propyn-1-ylthio derivatives.

Two new series of 1-(1,2,5-thiadiazol-4-yl)-4-azatricyclo[2.2.1.0(2, 6)]heptanes were synthesized and evaluated for their in vitro activity in cell lines transfected with either the human M1 or M2 receptor. 3-Phenyl-2-propyn-1-yloxy and -1-ylthio analogues substituted with halogen in the meta position showed high functional potency, efficacy, and selectivity toward the M1 receptor subtype. A quite unique functional M1 receptor selectivity was observed for compounds 8b, 8d, 8f, 9b, 9d, and 9f. Bioavailability studies in rats indicated an oral bioavailability of about 20-30%, with the N-oxide as the only detected metabolite.

[3H]-MK 912 binding delineates two alpha 2-adrenoceptor subtypes in rat CNS one of which is identical with the cloned pA2d alpha 2-adrenoceptor.

1. Simultaneous computer modelling of control and guanfacine-masked [3H]-MK 912 saturation curves as well as guanfacine competition curves revealed that the drugs bound to two alpha 2-adrenoceptor subtypes in the rat cerebral cortex with very different selectivities. These alpha 2-adrenoceptor subtypes were designated alpha 2A and alpha 2C. The Kd value of [3H]-MK 912 for the alpha 2A-subtype was 1.77 nM and for the alpha 2C-subtype 0.075 nM; the receptor sites showing capacities 296 and 33 fmol mg-1 protein, respectively. The Kds of guanfacine were 19.9 and 344 nM, respectively. 2. Binding constants of 26 compounds for the two rat cerebral cortex alpha 2-adrenoceptor subtypes were determined by simultaneous computer modelling of control and guanfacine-masked drug competition curves as well as plain guanfacine competition curves using [3H]-MK912 as labelled ligand (i.e. a '3-curve assay'). Of the tested drugs WB4101, corynanthine, rauwolscine, yohimbine, ARC 239 and prazosin were found to be clearly alpha 2C-selective with selectivities ranging from 16 to 30 fold whereas guanfacine, oxymetazoline, BRL 44408 and BRL 41992 were found to be alpha 2A-selective with selectivities ranging from 9 to 22 fold. 3. The Kds of compounds obtained for the cerebral cortex alpha 2C-adrenoceptors showed an almost 1:1 correlation with the corresponding Kds for alpha 2-adrenoceptors expressed by the pA2d-gene (the rat 'alpha 2-C4' adrenoceptor) in CHO-cells. The cerebral cortex alpha 2A-adrenoceptors did not correlate well with the pA2d alpha 2-adrenoceptor Kds. 4. In the rat spinal cord [3H]-MK 912 bound to alpha 2A- and alpha 2C-adrenoceptor sites with similar affinities as in the cerebral cortex and with densities 172 and 7.4 fmol mg-1 protein, respectively. Drug affinities for some compounds showing major selectivity for alpha 2A- and alpha 2C-adrenoceptors were fully compatible with the notion that the spinal cord sites were alpha 2A- and alpha 2C-adrenoceptors.

Independent induction of morphological transformation of CHO cells by receptor-activated cyclic AMP synthesis or by receptor-operated calcium influx.

Morphological transformation of Chinese hamster ovary (CHO) cells can be induced by exogenous addition of cyclic AMP (cAMP) or through the stimulation of G protein-coupled receptors ectopically expressed in these cells. The morphological transformation has been shown to represent a phenotypic suppression of CHO cell tumorigenic potential. Studies were undertaken to determine which receptor-activated signal transduction pathway initiates the progression from a tumorigenic to a non-tumorigenic phenotype. Stimulation of CHO cells expressing the dopamine D1 receptor (CHOD1) with a D1 selective agonist, SKF38393, resulted in an increase in cAMP accumulation which correlated with morphologic transformation. SKF38393 had no effect on intracellular calcium levels, arguing against a requirement for phospholipase C or calcium mobilization in the D1-stimulated morphology change. In contrast, stimulation of muscarinic m5 (CHOm5) or vasopressin V1a (CHOV1a) receptors expressed in CHO cells with carbachol or arginine vasopressin (AVP), respectively, did not result in an increase in intracellular calcium and a morphology change. The time course of carbachol-stimulated calcium influx correlated with the time course of morphological transformation, but not with carbachol-stimulated cAMP or inositol, 1,4,5-trisphosphate (IP3) accumulation. Furthermore, no increase in cAMP accumulation was observed in AVP-stimulated CHOV1a cells, suggesting a cAMP-independent stimulation of the transformation process. Carbachol-stimulated CHO cells expressing the m2 muscarinic receptor (CHOm2) failed to undergo a morphological transformation, yet released IP3. Therefore, phospholipase C-mediated signal transduction is not sufficient for the morphological transformation of CHO cells. It appears that receptor-stimulated morphologic transformation of CHO cells can be induced via two independent signaling pathways, mediated by adenylate cyclase or receptor-operated calcium channels.

Muscarinic receptor activated Ca2+ channels in non-excitable cells.

We have provided preliminary characterization of a single channel Ca2+ conductance in CHO cells. We have demonstrated that the channel conducts Ca2+, is regulated by m5 receptors, is voltage-independent, has an extremely low conductance, and is second messenger-independent. This channel may be the receptor-operated channel required for downstream activation of several signaling events. It is not known what other cell types express the channel or if it is one of a larger group of related channels. It seems likely that Ca2+ influx-dependent signaling pathways, activated by the muscarinic m5 receptor, would utilize a plasma membrane resident Ca2+ channel to provide a steady source of Ca2+ from outside the cell. The transient nature of IP3-activated increases in intracellular Ca2+ make it an unlikely source of the sustained Ca2+ rise required for phospholipase regulation. This is especially surprising, since levels of intracellular Ca2+ achieved from the release of intracellular Ca2+ stores can be at least one order of magnitude higher than those achieved from extracellular influx (Berridge, 1993). The phospholipase A2 and phospholipase D involved in muscarinic receptor-mediated signaling have not been purified or cloned. It is possible that receptor-activated and Ca2+ influx-dependent phospholipases are integral membrane proteins located adjacent to both receptors and channels. The phospholipases may also translocate to the membrane following activation where they would gain access to the continuous Ca2+ flow. Purification and cloning of this and other related channels should provide better insight into their role in cell signaling.

The signal transducer for the dopamine-1 regulated sodium transport in renal cortical brush border membrane vesicles.

We have reported the presence of dopamine-1 (DA-1) and dopamine-2 (DA-2) receptors in renal brush border and basolateral membranes. DA-1 agonists stimulate adenylate cyclase (AC) and phospholipase C (PLC) activity in both membranes. Moreover, the ability of a DA-1 agonist (fenoldopam) to stimulate PLC activity is independent of AC activity. A DA-2 agonist (LY171555) by itself was without effect and did not enhance the ability of the DA-1 agonist to stimulate PLC activity. The DA-1 but not DA-2 agonists inhibit Na+/H+ exchange activity in brush border membrane vesicles (BBMV) and Na+/K(+)-ATPase activity in basolateral membranes. However, cAMP inhibits, while protein kinase C (presumably via PLC activity) stimulates, Na+/H+ exchange activity. We therefore determined the effect of DA-1 agonists on Na+/H+ exchange activity when PLC or AC activity was blocked using neomycin or dideoxyadenosine, respectively. The drugs were incubated with minced renal cortex prior to preparation of BBMV by differential centrifugation and MnCl2 precipitation. Enrichment of BBMV was not affected by drug treatment. The Na+/H+ exchange activity was assessed by measuring amiloride (1 mmol/L) sensitive 22Na+ uptake in BBMV (pHi = 5.5, pHo = 7.5, Nai+ = O, Nao+ = 1 mmol/L). Neomycin inhibited DA and DA-1-stimulated PLC activity in BBMV in a concentration dependent manner (10(-6) to 10(-4) mol/L). Neomycin (10(-4) mol/L) completely blocked the ability of DA and DA-1 agonist to stimulate PLC activity but had no consistent effect on DA-1 inhibited Na+/H+ exchange activity. Dideoxyadenosine inhibited DA and DA-1 simulated AC activity without affecting DA-1 stimulated PLC activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Muscarinic acetylcholine receptor subtypes associated with release of Alzheimer amyloid precursor derivatives activate multiple signal transduction pathways.

Five subtypes of muscarinic acetylcholine receptors have been identified and designated m1-m5. The m1 and m3 receptors have recently been shown to stimulate APP processing. The m1 and m3 receptors couple to a variety of signal transduction pathways in both tissue slices and a variety of cell lines endogenously expressing either or both subtypes. In contrast, the m2 and m4 receptors have been primarily associated with inhibition of adenylate cyclase. We have transfected all five subtypes of muscarinic receptors into a variety of mammalian cell lines in order to investigate the signaling associated with single receptor subtypes. The m1, m3, or m5 receptors stimulate phospholipase A2, C, and D, adenylate cyclase, receptor-operated calcium channels, and tyrosine kinase activity simultaneously. The m2 or m4 receptor inhibits cAMP accumulation and augments a previously stimulated release of arachidonic acid and calcium influx.

Receptor-coupled amyloid precursor protein processing.

The family of beta-amyloid protein precursors (APP) can be processed via several alternative proteolytic pathways. Some generate potentially amyloidogenic APP derivatives, whereas others preclude the formation of such fragments. The cellular mechanisms regulating the relative activities of these pathways are thus important in determining the factors contributing to the formation of amyloidogenic APP derivatives. In order to investigate whether cell-surface receptor activity can regulate APP processing, HEK 293 cell lines stably expressing human muscarinic acetylcholine receptors (mAChR; subtypes m1, m2, m3, m4) were stimulated with the muscarinic agonist carbachol, and the release of APP derivatives was measured. Carbachol increased the release of large amino-terminal APP-fragments 4- to 6-fold in cell lines expressing the m1 or m3 receptors but not in those expressing m2 or m4 subtypes. This increase was blocked by various protein kinase inhibitors and mimicked by phorbol esters, indicating that it is mediated by protein kinase activation, presumably by protein kinase C (PKC). To determine whether additional cell-surface receptor types linked to this signal transduction pathway could also regulate APP processing, we stimulated differentiated PC-12 cells with bradykinin and found that this neuropeptide also increased the secretion of amino-terminal APP derivatives. We next investigated the possibility that neuronal depolarization might affect APP processing in mammalian brain. Electrically stimulated rat hippocampal slices released two times more amino-terminal APP derivatives than unstimulated control slices. This release increased with increasing stimulation frequencies in the physiological firing range of hippocampal pyramidal cells, and was blocked by tetrodotoxin. These results suggest that, in brain, APP processing is regulated by neuronal activity.

Dopamine D1 receptor regulation of phospholipase C.

Dopamine is an endogenous catecholamine which exerts its actions by occupancy of specific receptors. Dopamine receptors are classified into two main groups: the two cloned D1-like receptors (D1A and D1B in rats; D1B is also known as D5 in humans) are linked to stimulation of adenylyl cyclase, while the three cloned D2-like receptors (D2 or D2A, D3 or D2B, D4 or D2C) are linked to inhibition of adenylyl cyclase. All these dopamine receptors originally cloned from the brain are expressed in tissues outside the central nervous system including the kidney. Dopamine regulates many cellular activities, including transmembrane ion transport. Activation of D1-like receptor decreases sodium transport by cAMP dependent and cAMP independent mechanisms. Dopamine, via D1-like receptors, may inhibit Na+/H+ exchange activity in renal brush border membranes by a cAMP independent/Gs alpha-linked mechanism. Another cAMP independent pathway of sodium transport inhibition is mediated by phospholipase C, which has several isoforms (PLC beta, PLC gamma, and PLC delta with several members in each). Catecholamines stimulate expression and activity of phospholipase C isoforms in a concentration, time, and receptor-dependent as well as regional and subcellular compartmental-specific manner. In renal cortical membranes, intrarenal administration of norepinephrine for 3-4 h increases PLC beta expression and activity but has no effect on PLC gamma activity. In contrast, intrarenal administration of a D1 agonist for 3-4 h increases PLC beta 1 but decreases PLC gamma expression and activity.(ABSTRACT TRUNCATED AT 250 WORDS)