Characterization of the expression pattern of adrenergic receptors in rat taste buds.

Taste buds signal the presence of chemical stimuli in the oral cavity to the central nervous system using both early transduction mechanisms, which allow single cells to be depolarized via receptor-mediated signaling pathways, and late transduction mechanisms, which involve extensive cell-to-cell communication among the cells in the bud. The latter mechanisms, which involve a large number of neurotransmitters and neuropeptides, are less well understood. Among neurotransmitters, multiple lines of evidence suggest that norepinephrine plays a yet unknown role in the taste bud. This study investigated the expression pattern of adrenergic receptors in the rat posterior taste bud. Expression of alpha1A, alpha1B, alpha1D, alpha2A, alpha2B, alpha2C, beta1, and the beta2 adrenoceptor subtypes was observed in taste buds using RT-PCR and immunocytochemical techniques. Taste buds also expressed the biosynthetic enzyme for norepinephrine, dopamine beta-hydroxylase (DbetaH), as well as the norepinephrine transporter. Further, expression of the epinephrine synthetic enzyme, phenylethanolamine N-methyltransferase (PNMT), was observed suggesting a possible role for this transmitter in the bud. Phenotyping adrenoceptor expression patterns with double labeling experiments to gustducin, synaptosomal-associated protein 25 (SNAP-25), and neural cell adhesion molecule (NCAM) suggests they are prominently expressed in subsets of cells known to express taste receptor molecules but segregated from cells known to have synapses with the afferent nerve fiber. Alpha and beta adrenoceptors co-express with one another in unique patterns as observed with immunocytochemistry and single cell reverse transcription polymerase chain reaction (RT-PCR). These data suggest that single cells express multiple adrenergic receptors and that adrenergic signaling may be particularly important in bitter, sweet, and umami taste qualities. In summary, adrenergic signaling in the taste bud occurs through complex pathways that include presynaptic and postsynaptic receptors and likely play modulatory roles in processing of gustatory information similar to other peripheral sensory systems such as the retina, cochlea, and olfactory bulb.

Alpha-adrenoceptor agonistic activity of oxymetazoline and xylometazoline.

Oxymetazoline and xylometazoline are both used as nasal mucosa decongesting α-adrenoceptor agonists during a common cold. However, it is largely unknown which of the six α-adrenoceptor subtypes are actually present in human nasal mucosa, which are activated by the two alpha-adrenoceptor agonists and to what extent. Therefore, mRNA expression in human nasal mucosa of the six α-adrenoceptor subtypes was studied. Furthermore, the affinity and potency of the imidazolines oxymetazoline and xylometazoline at these α-adrenoceptor subtypes were examined in transfected HEK293 cells. The rank order of mRNA levels of α-adrenoceptor subtypes in human nasal mucosa was: α(2A) > α(1A) ≥ α(2B) > α(1D) ≥ α(2C) >> α(1B) . Oxymetazoline and xylometazoline exhibited in radioligand competition studies higher affinities than the catecholamines adrenaline and noradrenaline at most α-adrenoceptor subtypes. Compared to xylometazoline, oxymetazoline exhibited a significantly higher affinity at α(1A) - but a lower affinity at α(2B) -adrenoceptors. In functional studies in which adrenoceptor-mediated Ca(2+) signals were measured, both, oxymetazoline and xylometazoline behaved at α(2B) -adrenoceptors as full agonists but oxymetazoline was significantly more potent than xylometazoline. Furthermore, oxymetazoline was also a partial agonist at α(1A) -adrenoceptors; however, its potency was relatively low and it was much lower than its affinity. The higher potency at α(2B) -adrenoceptors, i.e. at receptors highly expressed at the mRNA level in human nasal mucosa, could eventually explain why in nasal decongestants oxymetazoline can be used in lower concentrations than xylometazoline.

Availability of saliva for the assessment of alterations in the autonomic nervous system caused by physical exercise training.

OBJECTIVE: The purpose of the study was to assess the alterations of the autonomic nervous system activities caused by physical exercise training under food restriction using a running wheel in mice based on an analysis of saliva. METHODS: Male ICR mice, weighing 39-50 g, at 32 weeks of age were divided into three groups; an exercise with food restriction (EXP), an exercise without food restriction (EXA) and a control (CTL). The EXP group was fed the same amount of diet as the CTL group (pair-feeding). The EXP and EXA groups used a "voluntary running wheel" for exercise. The pilocarpine stimulated whole saliva was collected from the oral cavity by micropipette over 15 min 4, 8 and 12 weeks after the beginning of the experiment. The salivary flow rate, protein concentration and amylase and kallikrein activities were determined, since amylase and kallikrein release have been shown to be evoked by beta- and alpha-adrenergic receptor stimulation, respectively. RESULTS: There was no significant difference in the sera corticosterone levels among the three experimental groups. The flow rate of saliva per total salivary gland weight in the EXP was significantly lower than that in the CTL and the EXA groups. The total protein secretion and kallikrein activity decreased by 20-30% in the EXP mice between 4 and 12 weeks after the starting of experiments in comparison to the CTL mice, thus suggesting that signal transduction in the alpha-sympathetic nervous system was downregulated. There was no significant difference in the amylase activity between the EXP and the CTL groups. CONCLUSION: Physical exercise under diet control induced alterations in saliva secretion, while exercise training alone did not affect the content of saliva. The results demonstrate the availability of saliva and suggest that the present experimental situation is a suitable experimental model for sports activity performed under diet control.

17-Beta-estradiol directly regulates the expression of adrenergic receptors and kisspeptin/GPR54 system in GT1-7 GnRH neurons.

Estradiol plays a critical role in the feedback regulation of reproduction, in part by modulating the neurosecretory activity of gonadotropin-releasing hormone (GnRH) neurons. While indirect effects of estradiol on GnRH neurons have been clearly demonstrated, direct actions are still controversial. In the current study, we examined direct effects of 17beta-estradiol upon the expression of receptors for afferent signals at the level of the GnRH neuron, using immortalized GT1-7 cells. Using RT-PCR, we confirmed the expression of mRNA for the adrenergic receptors (AR) alpha(1)A-, alpha(1)B-, alpha(1)D-, alpha(2)A-, alpha(2)C-, and beta(1)-AR, and showed for the first time that mRNAs for alpha(2)B-, beta(2)- and beta(3)-AR, for kisspeptin and its receptor GPR54 and for the novel estrogenic receptor GPR30 are expressed in GT1-7 cells. After treatment with 10 nM 17beta-estradiol, alpha(1)B-AR mRNA was significantly increased (14-fold) after 6 h as determined by real-time PCR, while alpha(1)B- and alpha(1)D-AR mRNA were significantly increased (19- and 23-fold, respectively) after 24 h. The expression of KiSS-1 and GPR54 mRNAs were also significantly increased (8- and 6-fold, respectively) after 24 h treatment of GT1-7 cells with estradiol. GPR30 mRNA expression was not affected by estradiol. Our data also showed that kisspeptin-10 (1-10 nM) can significantly stimulate GnRH release and GnRH mRNA expression in GT1-7 cells. These results suggest that the complex physiologic effects of estradiol on the function of the reproductive axis could be mediated partly through direct modulation of the expression of receptors for afferent signals in GnRH neurons.

Alpha-adrenergic mRNA subtype expression in the human nasal turbinate.

PURPOSE: Alpha-adrenergic receptor (AR) agonist drugs (e.g., epinephrine) are commonly used for upper airway procedures, to shrink the mucosa, retard absorption of local anesthetic agents, and improve visualization by limiting hemorrhage. Decongestant therapy often also includes alphaAR agonist agents, however overuse of these drugs (e.g., oxymetazoline) can result in chronic rhinitis and rebound increases in nasal secretion. Since current decongestants stimulate alphaARs non-selectively, characterization of alphaAR subtype distribution in human airway (nasal turbinate) offers an opportunity to refine therapeutic targets while minimizing side-effects. We, therefore, investigated alphaAR subtype expression in human nasal turbinate within epithelial, duct, gland, and vessel cells using in situ hybridization. METHODS: Since sensitive and specific anti-receptor antibodies and highly selective alphaAR subtype ligands are currently unavailable, in situ hybridization was performed on sections of three human nasal turbinate samples to identify distribution of alphaAR subtype mRNA. Subtype specific (35)S-labelled mRNA probes were incubated with nasal turbinate sections, and protected fragments remaining after RNase treatment analyzed by light and darkfield microscopy. RESULTS: In non-vascular tissue alpha(1d) AR mRNA predominates, whereas notably the alpha(2c) is the only alphaAR subtype present in the sinusoids and arteriovenous anastamoses. CONCLUSION: Combined with the current understanding that AR-mediated constriction of nasal sinusoids underpins decongestant therapies that minimize secretions and shrink tissues for airway procedures, these findings suggest that alpha(2c) AR subtypes provide a novel selective target for decongestant therapy in humans.

Balloon injury alters alpha-adrenoceptor expression across rat carotid artery wall.

alpha(1)-Adrenoceptors (AR) mediate growth factor-like activity of catecholamines on vascular smooth muscle cells (SMC) and adventitial fibroblasts. This trophic activity is strongly augmented by balloon injury, contributes significantly to subsequent proliferation, wall hypertrophy and lumen loss and is mediated by alpha(1A)- and alpha(1B)-AR. However, it is not known how injury augments adrenergic trophic activity. The aim of the present study was to examine alpha-AR expression in rat carotid artery and to test the hypothesis that balloon injury augments a(1)-AR expression. 2. Neointima, media and adventitia were isolated at various days after balloon injury of rat carotid artery and subjected to quantitative reverse transcription-polymerase chain reaction and radioligand binding. Cultured SMC were also studied. 3. Transcripts for alpha(1A)-, alpha(1B)-, alpha(1D)- and alpha(2D)-AR were expressed in different proportions in media and adventitia from uninjured carotid artery. Injury caused a reduction by as much as 85% at day 4 in all alpha-AR mRNA (but not cyclophilin) in both the media and adventitia. In both layers, expression returned to control by day 21 for alpha(2D)-AR and by day 42 for alpha(1A)-AR, but remained reduced by 25-50% for alpha(1B)- and alpha(1D)-AR at 42 days. alpha(1)-Adrenoceptor transcripts in the neointima at 21 and 42 days after injury were expressed at levels more than 80% lower than in the media or adventitia of uninjured carotid; alpha(2D)-AR mRNA was undetectable. The density of total alpha(1)-AR binding sites was similar in the media and adventitia of uninjured carotid. Density was reduced by approximately 60% in the intima-media and adventitia 21 days after injury. To examine possible mechanisms, early passaged cultured SMC were studied that express alpha(1D)- and alpha(1B)-AR at levels similar to in vivo but that do not express other alpha-AR. Basic fibroblast growth factor caused downregulation of alpha(1D)-AR mRNA and alpha(1)-AR density, without affecting mRNA half-life, whereas transforming growth factor-beta1 had no effect. Neither growth factor altered alpha(1B)-AR message expression. 4. These data demonstrate that: (i) carotid artery expresses the same four alpha-AR genes and similar total alpha(1)-AR density in the SMC media and fibroblast-rich adventitia; and (ii) injury induced enhancement of adrenergic trophic activity is not caused by upregulation of alpha(1)-AR, but, instead, is associated with a generalized reduction in alpha-AR expression.

Ontogenesis of mRNA levels and binding sites of hepatic alpha-adrenoceptors in young cattle.

Catecholamines affect hepatic glucose production through (alpha- and beta2-) adrenoceptors (AR). We studied mRNA abundance and binding of hepatic alpha-AR in pre-term (P0) calves and in full-term calves at day 0 (F0), day 5 (F5) and day 159 (F159) to test the hypothesis that gene expression and numbers of hepatic alpha-AR in calves are influenced by age and associated with beta2-AR and selected traits of glucose metabolism. mRNA levels of alpha1- and alpha2-AR were measured by real time RT-PCR. alpha1- and alpha2-AR numbers (maximal binding, Bmax) were determined by saturation binding of (3H)-prazosin and (3H)-RX821002, respectively. alpha1- and alpha2-AR subtypes were evaluated by competitive binding. alpha1A-AR mRNA levels were lower in P0 than in F0, F5 and F159 and alpha(2AD)-AR mRNA levels were lower in F159 than in P0, F0 and F5, while alpha2C-AR mRNA levels increased from P0 and F0 to F5 and F159. Bmax of alpha1-AR increased from P0 to F5, then decreased in F159. Bmax of alpha2-AR decreased from F0 to F159. Bmax of alpha1-AR was positively associated with mRNA levels of alpha1A-AR (r = 0.7), Bmax of beta2-AR (r = 0.5) and negatively with hepatic glycogen content (r = -0.6). Bmax of alpha2-AR was negatively associated with Bmax of beta2-AR (r = -0.4). In conclusion, mRNA levels and binding sites of alpha1- and alpha2-AR in calves exhibited developmental changes and were negatively associated with hepatic glycogen content.

Cytokines down-regulate alpha1-adrenergic receptor expression during endotoxemia.

OBJECTIVE: The reduced pressure response to norepinephrine in septic patients has directed our interest to the regulation of alpha1-adrenergic receptors in vitro and in vivo during conditions mimicking acute sepsis. DESIGN: Prospective animal trial followed by a controlled cell culture study. SETTING: Laboratory of the Department of Anesthesiology. SUBJECTS: Male Sprague-Dawley rats weighing 200 to 250 g and a mesangial cell line. INTERVENTIONS: Experimental endotoxemia was induced in rats with lipopolysaccharide, and blood pressure dose-response studies with norepinephrine were performed. Alpha1-receptor gene expression was determined in various organs by a specific RNase protection assay, and tissue concentrations of the proinflammatory cytokines interleukin-1beta and tumor necrosis factor-alpha were measured. Rat renal mesangial cells were incubated with these cytokines or with nitric oxide donors to investigate the regulation of alpha1-adrenergic receptors during severe inflammation on a cellular level. MEASUREMENTS AND MAIN RESULTS: The pressor effect of norepinephrine was markedly diminished during endotoxemia. The animals showed down-regulated mRNA levels of alpha1A-, alpha1B- and alpha1D-receptors in all organs investigated, and the tissue concentrations of interleukin-1beta and tumor necrosis factor-alpha were highly increased during experimental endotoxemia. Incubation of cultured rat renal mesangial cells with the cytokines resulted in diminished alpha -receptor gene expression and [3H]prazosin binding capacity, whereas incubation of the cells with nitric oxide donors did not affect alpha1B-receptor expression. In line, blocking of cytokine-induced nitric oxide synthesis by coincubation of mesangial cells with N(G)-nitro-L-arginine methyl ester did not influence cytokine-induced down-regulation of alpha1B-receptors. CONCLUSIONS: Our data show that endotoxemia causes a systemic down-regulation of alpha1-receptors on the level of gene expression and suggest that this effect is likely mediated by proinflammatory cytokines in a synergistic but nitric oxide-independent fashion. We propose that this down-regulation of alpha1-adrenergic receptors contributes to the attenuated blood pressure response to norepinephrine and, therefore, to septic circulatory failure in patients.

Expression of alpha-adrenoceptor subtypes by smooth muscle cells and adventitial fibroblasts in rat aorta and in cell culture.

Previous radioligand binding reports of vascular alpha-adrenoceptor (AR) density have been limited to total alpha1- or alpha2-ARs. Studies using whole blood vessel homogenates have not differentiated among receptor or mRNA expression by medial smooth muscle cells (SMCs) versus adventitial fibroblasts (AFBs). Therefore, we used quantitative reverse transcription-polymerase chain reaction and radioligand binding to measure alpha-AR subtypes in media, adventitia, and cultured SMCs and AFBs from rat aorta. Both media and adventitia expressed alpha1A-, alpha1B-, alpha1D-, and alpha2D-AR mRNAs, but in markedly different abundances. Total alpha1-AR density was the same for media and adventitia (Bmax = 101 +/- 10 versus 96 +/- 16 fmol/mg of protein). However, densities for alpha1A-, alpha1B-, and alpha1D-AR subtypes in media were 19 +/- 2, 26 +/- 4, and 55 +/- 2%, and in adventitia were 44 +/- 3, 37 +/- 5, and 19 +/- 2%. No alpha2B- or alpha2C-AR transcripts were detected in either layer or in cultured SMCs or AFBs. Total alpha1-AR densities in cultured SMCs and AFBs (Bmax = 111 +/- 4 and 48 +/- 6 fmol/mg of protein, respectively) were similar to media and adventitia, with alpha1B- and alpha1D-AR transcript levels and receptors largely sustained. However, alpha1A- and alpha2D-AR expression in cultured SMCs and AFBs was strongly reduced, compared with media and adventitia, an effect not prevented by 30 different culture conditions. Like SMCs, exposure of AFBs to norepinephrine induced protein synthesis and proliferation of AFBs. This is the first study to quantitate alpha-AR subtype expression in media and adventitia and in cultured SMCs and AFBs. In addition, we report the intriguing finding that AFBs express alpha1-ARs in similar abundance as medial SMCs and that norepinephrine induced them to proliferate.

Norepinephrine-induced migration of SW 480 colon carcinoma cells is inhibited by beta-blockers.

Beta-adrenoceptors are highly expressed on SW 480 colon carcinoma cells as was assessed by flow cytometry. We investigated the influence of norepinephrine on the migration of these cells using time-lapse videomicroscopy. Norepinephrine-treatment increased the locomotor activity within the population from 25% spontaneously locomoting cells to 65% locomoting cells. The beta1/2-blocker propranolol but not the beta1-blocker atenolol inhibited this increase. The intracellular signaling solely of norepinephrine-induced locomotion involved protein tyrosine kinase activity, whereas both spontaneous and norepinephrine-induced migration were reduced by inhibiting phospholipase Cgamma and protein kinase Calpha activity. In summary, norepinephrine-induced locomotion of SW 480 cells is beta2-adrenoceptor mediated and distinct from spontaneous locomotion concerning the PTK involvement.

Expression of multiple alpha-adrenoceptor isoforms in rat CCD.

In the rat cortical collecting duct (CCD), epinephrine inhibits vasopressin (AVP)-dependent water permeability and Na+ reabsorption. Although inhibition is reversed by the alpha2-adrenoceptor (AR) antagonist yohimbine, suggesting the epinephrine effect is primarily mediated by an alpha2-AR [C. T. Hawk, L. H. Kudo, A. J. Rouch, and J. A. Schafer. Am. J. Physiol. 265 (Renal Fluid Electrolyte Physiol. 34): F449-F460, 1993], there are also suggestions of an effect at an additional receptor, perhaps an alpha1-AR. For the present experiments, we used RT-PCR of total RNA extracted from 1 to 5 mm of microdissected CCDs from rat kidney to identify the alpha-AR isoforms expressed. Specific primers for the alpha2-ARs amplifying from the 6th transmembrane (TM) to the 3'-untranslated regions, revealed the presence of alpha2A and alpha2B. Western blot analysis also indicated the presence of alpha2B-AR at the protein level. Degenerate alpha1-AR primers that amplify from conserved regions of TM-1 to TM-5, as well as specific primers that amplify either the same region (alpha1B), the carboxy terminus (alpha1A), or within the third cytoplasmic loop (alpha1D), indicated the presence of all three alpha1-ARs. Measurement of transepithelial voltage in isolated perfused renal tubules indicated a small inhibitory effect mediated by alpha1-ARs. Although the functional effects of epinephrine on AVP-dependent transport processes appear to be mediated predominantly by an alpha2-AR, a small contribution to the overall alpha-AR effect may be due to simultaneous activation of an alpha1-AR.

A novel aortic smooth muscle cell line obtained from p53 knock out mice expresses several differentiation characteristics.

Here we report that we could obtain a highly differentiated smooth muscle cell line by screening the expression of a-smooth muscle actin from p53 knook out mice aorta. This cell revealed extended bipolar shape and expressed h-caldesmon and calponin as well as a-smooth muscle actin as protein markers of differentiated smooth muscle. Further intracellular calcium increase was induced by application of noradrenaline in a dose dependent manner and calcium oscillation was also observed in a higher dose (100 microM). Appropriate application of 5-azacytidine enhanced these tendencies and induced slow contraction by endothelin-1 and phenylephrine.

Transient expression of alpha-1B adrenoceptor messenger ribonucleic acids in the rat superior cervical ganglion during postnatal development.

We have examined the developmental profile of the alpha-1 and alpha-2 adrenergic receptor messenger ribonucleic acids expression in the rat superior cervical ganglion. The expression of the six messenger ribonucleic acids was studied using reverse transcription-polymerase chain reaction. At four weeks, the dominant messenger ribonucleic acids transcripts in this sympathetic ganglion were alpha-1C, alpha-2A, alpha-2B and alpha-2C. The expression of alpha-1 genes in the superior cervical ganglion appears to be regulated during postnatal development in that two alpha-1 (alpha-1B, alpha-1C) genes were expressed at birth, three, seven and 14 days postnatal but no amplified product for alpha-1B was detected at 28 days and in the aged animals, while the alpha-1C transcript continued to be expressed. No amplified product for alpha-1D was detected in superior cervical ganglion at any of the ages studied. While all three alpha-2 genes were expressed in the superior cervical ganglion at four weeks the dominant alpha-2 messenger ribonucleic acids transcript expressed in the superior cervical ganglion was alpha-2A. This pattern of alpha-2 adrenoceptor gene expression was maintained from birth, throughout development and into old age. These results suggest that the expression of alpha-1 adrenergic receptors in the superior cervical ganglion is regulated developmentally while the expression of alpha-2 genes remains unchanged.

Cardiac alpha-adrenergic receptor expression is regulated by thyroid hormone during a critical developmental period.

Although thyroid hormone is obligatory for the development of cardiac beta-adrenergic receptors, it is difficult to assign a specific role for the hormone in receptor ontogeny because beta-receptor expression is affected similarly in the adult. We have determined whether thyroid hormone plays a role in receptor development by evaluating alpha 1-adrenergic receptors, which in the adult are downregulated by thyroid hormone. Propylthiouracil given from gestational day 17 through postnatal day 5 caused significant deficits in the number of alpha 1-receptors and values resolved to normal in parallel with hormone level recovery. When propylthiouracil was administered later (postnatal days 11 through 15) only a transient deficit in alpha 1-receptor binding was seen; hyperthyroidism (triiodothyronine) could still evoke stimulation of receptor expression at this stage. The effects on receptor expression were distinguished from general effects on cell differentiation by examining alpha 2-receptors, which disappear over the first three postnatal weeks; delayed differentiation caused by propylthiouracil would slow the decline in alpha 2-receptors, whereas accelerated differentiation caused by triiodothyronine would hasten the decline. Instead, the effects were similar to those on alpha 1-receptors: perinatal propylthiouracil administration reduced, and neonatal triiodothyronine administration enhanced, alpha 2-receptor binding sites. Thus, thyroid hormone plays a role in the control of cardiac adrenergic receptor expression during a critical development period, with conjoint regulation of the multiple receptor subtypes present within the tissue. As adrenergic stimulation is important in maintaining cardiac function in the perinatal period, alterations of thyroid status during this period can be expected to result in abnormal reactivity and increased perinatal risk.

Alpha-adrenoceptors and angiotensinogen gene expression in opossum kidney cells.

To investigate whether alpha (alpha)-adrenoceptor agonists have a stimulatory effect on the expression of the angiotensinogen (Ang) gene in opossum kidney (OK) cells, we used OK 27 cells with a fusion gene containing the 5'-flanking regulatory sequence of the rat angiotensinogen gene fused with a human growth hormone (hGH) gene as a reporter, pOGH (Ang N-1498/+18), permanently integrated into their genomes. The level of expression of the pOGH (Ang N-1498/+18) was quantitated by the amount of immunoreactive-human growth hormone (IR-hGH) secreted into the medium. The addition of iodoclonidine (alpha 2-adrenoceptor agonist, 10(-13) to 10(-9) M) and phorbol 12-myristate 13-acetate (PMA, 10(-13) to 10(-5) M) stimulated the expression of pOGH (Ang N-1498/+18) in a dose-dependent manner, whereas the addition of phenylephrine (alpha 1-adrenoceptor agonist, 10(-13) to 10(-5) M) had no effect. The stimulatory effect of iodoclonidine was blocked by the presence of yohimbine (alpha 2-adrenoceptor antagonist) and staurosporine (an inhibitor of protein kinase C) but not blocked by the presence of prazosin (alpha 1-adrenoceptor antagonist) or Rp-cAMP (an inhibitor of cAMP-dependent protein kinase A). The addition of iodoclonidine, phenylephrine or PMA had no effect on the expression of pTKGH in OK 13 cells, an OK cell line, into which had been stably integrated a fusion gene, pTKGH containing the promoter/enhancer DNA sequence of the viral thymidine-kinase (TK) gene fused with a human growth hormone gene as a reporter.(ABSTRACT TRUNCATED AT 250 WORDS)

Cloning and expression of the alpha 2C-adrenergic receptor from the OK cell line.

The alpha 2-adrenergic receptors have been divided into four pharmacological subtypes, alpha 2A, alpha 2B, alpha 2C, and alpha 2D. The OK cell line, a cell line derived from an opossum kidney, expresses the alpha 2C-adrenergic receptor and is the prototypical cell line for the alpha 2C receptor subtype. The cloned human alpha 2C-C4 and rat RG10 receptors have been shown to express alpha 2C pharmacology. Here we report the cloning and expression of the OK alpha 2C-adrenergic receptor, OKc2. The receptor has 64% deduced amino acid identity and 21% similarity to the alpha 2-C4 receptor, giving an overall similarity of 85%. The clone, expressed in Chinese hamster ovary cells, has a pharmacology that correlates very well (r = 0.97) with that of the native OK cell alpha 2C-adrenergic receptor, and it is negatively coupled to adenylyl cyclase.

Cloning, functional expression and tissue distribution of human cDNA for the alpha 1C-adrenergic receptor.

We have cloned human alpha 1C-adrenergic receptor from human prostate cDNA library. The deduced amino acid sequence of the clone (P2C4) encodes a protein of 466 amino acids that showed strong sequence homology to the previously cloned bovine alpha 1C-adrenergic receptor. The radioligand binding properties of P2C4 expressed in COS-7 cells were very similar to those of bovine alpha 1C-adrenergic receptor. With reverse-transcription polymerase chain reaction assay, we observed alpha 1C-adrenergic receptor transcripts in heart, brain, liver and prostate, but not in kidney, lung, adrenal, aorta and pituitary. The data show that the clone P2C4 encodes a human alpha 1C-adrenergic receptor cDNA, and the receptor subtype is expressed not widely but localized in several human tissues.

Receptor-evoked Cl- current in Xenopus oocytes is mediated through a beta-type phospholipase C. Cloning of a new form of the enzyme.

Xenopus oocytes exhibit a receptor-evoked Cl- current that is mediated through the activation of phospholipase C (PLC) and release of intracellular Ca2+. The identity of PLC(s) mediating this effect is unknown. We have cloned cDNAs encoding a new form of PLC-beta from a Xenopus oocyte cDNA library. The Xenopus PLC-beta has substantial (33-64%) homology with mammalian beta 1, beta 2, beta 3, and beta 4 phospholipase C and is closest to PLC-beta 3, with 64% identity and 80% similarity. Injection of antisense oligonucleotides to a specific region of Xenopus PLC-beta results in degradation of its mRNA and significantly reduces Cl- currents evoked by both endogenous angiotensin receptors and expressed mammalian alpha 1b-adrenergic receptors and M1-muscarinic receptors as compared to responses in sense oligonucleotide-injected oocytes. Inhibition of the M1-muscarinic response by antisense oligonucleotides was nonadditive with pertussis toxin inhibition. PLC antisense oligonucleotide-injected oocytes show Cl- current responses to IP3 that are indistinguishable from sense oligonucleotide-injected oocytes. Since the receptor responses are pertussis toxin-sensitive, we conclude that we have isolated a new form of PLC-beta involved in the pertussis toxin-sensitive receptor stimulation of the Ca2+ activated Cl- current in Xenopus oocytes.