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Query: UNIPROT:P56851 (epididymal)
 11,273 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Adrenergic regulation of epididymal Cl- currents was studied by the whole-cell patch-clamp technique using various alpha- and beta-receptor agonists and antagonists in primary cultured rat cauda epididymal cells. Cl- currents could be activated with varying frequency by noradrenaline (primarily alpha- and beta 1-adrenoceptor-selective agonist, 1-5 microM), isoprenaline (nonselective beta-adrenoceptor agonist, 5 microM), salbutamol (beta 2-adrenoceptor-selective agonist, 2 microM), and phenylephrine (alpha 1-adrenoceptor-selective agonist, 1-2 microM). Noradrenaline alone elicited Cl- current activation in 85% of the cells examined. In the presence of phentolamine (nonselective alpha-adrenoceptor antagonist, 15 microM), noradrenaline elicited Cl- current activation in 63% of the cells examined, whereas noradrenaline-induced activation was observed in 33% of the cells examined in the presence of both atenolol and butoxamine (beta 1- and beta 2-adrenoceptor antagonists, respectively, 10 microM). In 27% of single cells examined, a second current activation in response to salbutamol was observed after the first response to phenylephrine. When the order of stimuli was reversed, dual activation was also observed in 22% of the single cells examined, indicating the presence of both alpha- and beta-adrenoceptors in single epididymal cells. Profiles of time- and voltage-dependent Cl- current upon activation by different adrenoceptor agonists exhibited characteristics similar to those previously reported for Ca2+ and cAMP-activated Cl- currents, suggesting that regulation of epididymal Cl- conductances could be mediated by different adrenoceptor subtypes involving Ca2+ and cAMP as intracellular second messengers.
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PMID:Adrenergic receptors on cultured rat epididymal cells: regulation of Cl- conductances. 784 80

The biochemical locus of the decrease of lipolytic responsiveness to catecholamines in the aging rat has not heretofore been completely identified. Although increased sensitivity to the inhibitory action of adenosine is the likely explanation for the decrease during maturation, the nature of the age effect during senescence has been unclear. In order to determine whether the proximal or distal portion of the lipolytic pathway is involved, we have studied the lipolytic effect of the distally acting cyclic AMP analogue, 8-(4-chlorophenylthioadenosine)3'5'-monophosphate (cyclic) (Cl-cAMP) on rat fat cells from both the epididymal and perirenal fat pads of mature (6 mo) and senescent (24 mo) Fischer 344 rats. Using an adenosine (N6-1-2-phenylisopropyl-adenosine; PIA) regulated system, the lipolytic response to epinephrine (glycerol release) was measured simultaneously with that to Cl-cAMP. The effects of age on lipolysis are greatly influenced by the anatomic site of origin of the fat cells. The epididymal cells of the old rats showed no decreased responsiveness to either epinephrine or Cl-cAMP. However, the perirenal cells of the old rats showed a grossly impaired maximal response to both epinephrine (60% decrease relative to young; p < .005) and Cl-cAMP (42% and 58% decrease in 2 sets of experiments; p < .05 and .04, respectively). Although decreased lipolytic response to epinephrine in epididymal cells was not seen in these studies, this has been clearly shown in earlier work, suggesting that diminished response to epinephrine is demonstrable only when the system is not already maximally inhibited by PIA.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Catecholamine-sensitive lipolysis in the rat: different loci for effect of age on the lipolytic cascade in epididymal vs perirenal fat cells. 801 85

A study was carried out to investigate the short-circuit current (Isc) response to noradrenaline (NA) and the signal transduction mechanisms involved in cultured rat cauda epididymal epithelium. In normal Krebs-Henseleit solution, NA (10 mumol.l-1) added basolaterally elicited a biphasic Isc response consisting of a transient spike followed by a second sustained response. The biphasic response was almost abolished by removing ambient Cl-. Preloading the tissues with a cell-permeant Ca2+ chelator, 1,2-bis(2-aminophenoxy) ethane-N,N,N',N',-tetraacetic acid acetoxymethyl ester (BAPTA/AM), or pretreating them with thapsigargin (Tg), a microsomal adenosine triphosphatase inhibitor abolished the initial spike in the Isc response to NA, but had little effect on the second component. Pretreating the tissues with a non-selective beta-antagonist, nadolol, reduced the second Isc response in a dose-dependent fashion but the initial spike was not affected. Microfluorimetric studies showed that NA (100 mumol.l-1) elicited single Ca2+ spikes in isolated epididymal cells, which could be abolished by prior treatment with Tg. Biochemical assays showed that NA (10 mumol.l-1) increased intracellular cyclic adenosine monophosphate concentration ([cAMP]i) and the response was abolished by prior treatment with nadolol (50 mumol.l-1). The results showed that NA elicited a biphasic Isc response mediated by a rise in intracellular Ca2+ concentration ([Ca2+]i) followed by a rise in [cAMP]i. The Ca(2+)-mediated Isc response had a faster onset and more transient action than the cAMP counterpart. It is suggested that NA released from noradrenergic nerve endings regulates transepithelial Cl- secretion in the epididymis thereby providing the specialized millieu vital for sperm storage and maturation.
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PMID:Biphasic short-circuit current response to noradrenaline mediated by Ca2+ and cAMP in cultured rat epididymal epithelium. 801 89

Based upon recent reports that the mRNA from the regulatory (R) RI beta subunit of cAMP-dependent protein kinase (PKA) was expressed in testicular extracts, we determined whether testicular extracts exhibited RI beta protein. To accomplish this goal, we initially determined the fundamental labeling and ionic characteristics of recombinant RI beta. Recombinant RI beta eluted from DEAE-cellulose with a salt concentration (of 0.075 M) equivalent to its elution position from soluble mouse brain extracts with catalytic subunit-free RI alpha. As predicted by its amino acid sequence homology to RI alpha, recombinant RI beta was not phosphorylated by PKA but was labeled specifically with 8-azido-adenosine 3':5'-[32P]monophosphate (8-N3[32P]cAMP). Additionally, RI antisera reacted equally with RI alpha (47 kDa) and recombinant RI beta (53 kDa). However, recombinant RI beta exhibited an unexpectedly basic pI of 6.65-6.85. By using a pH gradient for isoelectric focussing that allowed for clear focussing of 8-N3[32P]cAMP-labeled recombinant RI beta, 8-N3[32P]cAMP-labeled RI beta was readily detected by two-dimensional gel electrophoresis in rat brain particulate extracts and exhibited a pI equivalent to that of recombinant RI beta. The 53-kDa RI beta was undetectable either by its immunoreactivity or upon photoaffinity labeling with 8-N3[32P]cAMP by one or two-dimensional gel electrophoresis in soluble or particulate extracts of testes of 14-day-old, 45-day-old, or adult rats or in epididymal sperm. However, 8-N3[32P]cAMP-labeled RI beta was detected, albeit in very small levels, by two-dimensional electrophoresis upon separation of PKAs in testes of 14-day-old rats by DEAE-cellulose chromatography but was absent in equivalent extracts from adult rat testes. These results demonstrate that the unexpectedly basic pI of RI beta allows for its clear separation by two-dimensional electrophoresis from the RII proteins and therefore allows for its unambiguous identification. Further studies, however, are required to resolve the basis for the apparent disparity in testis RI beta mRNA and protein.
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PMID:Characterization of recombinant RI beta and evaluation of the presence of RI beta protein in rat brain and testicular extracts. 803 21

Primary cultured rat efferent ductal epithelia and cauda epididymal epithelial were mounted in Ussing chambers to study the effect of arginine vasopressin (AVP) on chloride secretion in the male excurrent duct. The regional differences in the signal transduction pathways involved were also investigated. In both the efferent duct and the cauda epididymidis, basolateral addition of AVP resulted in a dose-dependent increase in the short-circuit current (Isc), which was mediated via V1 receptor. Replacement of ambient Cl- with gluconate or pretreatment of a Cl- channel blocker, diphenylamine-2-carboxylate (apical, 1 mM), completely abolished the response, whereas addition of amiloride had no effect on the Isc. Pretreating the epithelia of the efferent duct with indomethacin (apical, 5 microM) or forskolin (basolateral, 1 microM), but not thapsigargin (apical, 1 microM) or trifluoperazine (apical, 20 microM), significantly inhibited the AVP response (P < 0.001). By comparison, pretreating the epithelia of the cauda epididymidis with any of the four agents significantly reduced the AVP-evoked response. These results suggested that the stimulation of chloride secretion by AVP in the efferent duct and the cauda epididymidis is mediated by prostaglandin synthesis and involves adenosine 3',5'-cyclic monophosphate (cAMP) as a second messenger. In the cauda epididymidis, calcium, in addition to cAMP, may play a role in mediating the AVP-induced response.
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PMID:The effect of [Arg8]vasopressin on electrogenic chloride secretion in cultured rat epididymal epithelia. 807 93

Since cAMP is considered to play a major role in the acquisition of maturation and fertilizing capacity of mammalian sperm, we investigated the expression of cAMP-synthesizing adenylyl cyclase (AC) in sperm retrieved directly from the human epididymis. Particulate fractions were prepared from purified epididymal sperm samples and AC was monitored by the direct conversion of ATP into cAMP. We report that in great contrast to human ejaculated sperm and other mammalian sperm cells, the human epididymal sperm do not express a Mn(2+)-sensitive AC. However, a functional AC was readily detectable in these sperm cells in the presence of saturating concentrations of Ca2+ (50mM) and bicarbonate (HCO3-, 50mM), a combination that causes maximal activation in human ejaculated sperm. Using these conditions, human epididymal sperm AC showed similar capacity to generate cAMP compared to human ejaculated sperm AC. When assays were performed in the presence of Mg2+ and a saturating concentration of GMP-P(NH)P (50 microM), the hydrolysis-resistant GTP analog, and forskolin (100 microM), no activity was detected indicating that the epididymal sperm AC differs from that in somatic cells. These data demonstrate that human epididymal sperm contain an AC that is unique and different from the enzyme system described in somatic cells and other mammalian sperm cells, including human ejaculated sperm.
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PMID:Evidence for a novel adenylyl cyclase in human epididymal sperm. 824 32

Cyclic AMP-dependent changes in phosphorylation of epididymal mouse sperm suspensions were examined in media designed to manipulate capacitation and the expression of parameters associated with full fertilizing ability, i.e. hyperactivated motility and the acrosome reaction. After initial assessment of cAMP-dependent protein kinase activity in frozen-thawed and lyophilized sperm suspensions using exogenous substrate, phosphorylation of endogenous sperm phosphoproteins was examined using sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by autoradiography or immunoblotting. Numerous phosphoproteins were detected in both incapacitated and capacitated suspensions, the majority of which were probably concerned with motility; full expression of fertilizing ability appeared to involve an increase in the amount of endogenous phosphorylation as deduced from the decreased amount of 32P incorporation in these suspensions. The addition of the cAMP-dependent protein kinase inhibitors, H8 and PKI (6-22) amide, demonstrated that most of the phosphoproteins detected were phosphorylated in a cAMP-dependent manner. Of particular interest was a phosphoprotein with an M(r) of about 95,000 which was consistently observed in capacitated suspensions. Evidence suggests that this may be phosphorylated on tyrosine residues, since the inclusion of orthovanadate, a phosphoryltyrosine phosphatase inhibitor, altered phosphorylation of this protein. Furthermore, immunodetection using the antiphosphotyrosine antibody, PY-20, identified five proteins with approximate M(r) 116,000, 105,000, 95,000, 86,000, and 76,000, and possibly a sixth at 54,000. The 95,000 protein was consistently diminished in ionophore-treated spermatozoa, indicating that the protein was located in the acrosomal cap region. These results suggest that the protein may be the same phosphotyrosine-containing protein as that described by Leyton and Saling (1989) which has been proposed to play a role in acrosomal exocytosis.
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PMID:Cyclic AMP-dependent phosphorylation of epididymal mouse sperm proteins during capacitation in vitro: identification of an M(r) 95,000 phosphotyrosine-containing protein. 838 23

Multiple physiological functions have been described to be affected by adenosine in numerous cell types. A comparative study of the expression of adenosine receptors has been performed in preadipocytes and adipocytes from rat epididymal fat pad. The results show that, in agreement with its well known antilipolytic effect, adenosine induces a negative modulation of adenylate cyclase via the A1 receptor present in adipocytes. By contrast, the A2 receptor subtype, which is positively coupled to adenylate cyclase, is herein demonstrated to be only expressed in adipose precursor cells. This expression allows, in chemically defined medium, the adenosine analogue NECA, by means of its ability to elevate cAMP concentration, to potentiate differentiation. These findings emphasize the role that adenosine might play as a bimodal regulatory extracellular signal in adipose tissue development.
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PMID:Differential expression of adenosine A1 and A2 receptors in preadipocytes and adipocytes. 839 1

The activity of adipose tissue hormone-sensitive lipase in animals with hyperinsulinemia has been reported to be increased compared with that in control animals. We examined whether this results from a direct effect of insulin on the tissue and whether it is accompanied by alteration in the regulation of lipolysis. When rat epididymal fat pads are incubated in culture medium with bovine serum albumin for 2-4 h with 2 ng/ml or 50 microU/ml of insulin, hormone-sensitive lipase activity in the postmicrosomal supernatant fraction after acid precipitation and activation with ATP-Mg2+ increases significantly compared with preparations from tissues incubated with the vehicle. The specific activities of hormone-sensitive lipase in sonicates of adipocytes after primary culture with insulin at concentrations from 10 to 4000 ng/ml (250 microU to 100 mU/ml) increase in an insulin-dose-related manner. Lipolysis in response to 10(-7) M isoproterenol also increases in an insulin-dose-dependent manner. Enhancement of isoproterenol-mediated lipolysis is not attributable to a difference in the triglyceride content of the cells. Lipolysis caused by the beta-agonist could be completely blocked by the simultaneous presence of insulin in both control and insulin-treated cells reflecting normal responsiveness of both types of cells to the acute effect of insulin. Although an increase in lipolysis is seen with norepinephrine and growth hormone after insulin treatment, other lipolytic agents such as ACTH, thyrotropin, and glucagon evoke similar responses in insulin-treated and control cells. The simultaneous presence of growth hormone and insulin during the 16-h culture results in additive effects on the subsequent response of the cells to 10(-7) M isoproterenol compared with the responses of the cells cultured with each hormone alone. beta-Agonist-mediated cAMP accumulation in the presence of Ro-20.1724, a specific phosphodiesterase inhibitor, is significantly higher in cells cultured in the presence of insulin than in control cells. Forskolin (1-25 microM) increases the lipolytic responses of insulin-treated cells compared with control cells, but the maximal response of the insulin-treated cells to forskolin is lower than that to isoproterenol. We conclude that changes produced by chronic insulin treatment involve more than one site along the lipolytic cascade.
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PMID:Chronic exposure of rat fat cells to insulin enhances lipolysis and activation of partially purified hormone-sensitive lipase. 839 27

Activation of Ca2+ and cAMP-dependent Cl- conductances by extracellular ATP was studied using the whole-cell patch clamp technique. Immediately after addition of extracellular ATP (10 microM), activation of whole-cell Cl- current exhibiting delayed inactivation and activation kinetics at hyperpolarizing and depolarizing voltages, respectively, was observed. After prolonged activation, the kinetic characteristics of the ATP-induced Cl- current became time- and voltage-independent. When applied to the later phase of the ATP-activated whole-cell current, the disulfonic acid stilbene DIDS (200 microM) could only inhibit 64% of the current while diphenylamine-dicarboxylic acid (DPC, 1 mM) completely inhibited it. Inclusion of a peptide inhibitor for protein kinase A (PKI, 10 nM) in the pipette solution blocked ATP-induced time- and voltage-independent current activation but did not affect the delayed activating and inactivating current activation but did not affect the delayed activating and inactivating current which could be totally blocked by DIDS. Anion selectivity sequence was determined in the presence of either PKI or DIDS and found to be significantly different. Increased pipette EGTA (10 mM) or treatment of the cells with trifluoperazine (40 microM), an inhibitor of calmodulin, suppressed both types of ATP-induced Cl- currents. No current activation by ATP was observed when cells were dialyzed with the IP3 receptor blocker, heparin (10 ng/ml). These results suggest that extracellular ATP activates IP3-linked Ca(2+)-dependent regulatory pathway, which in turn activates cAMP-dependent pathway, leading to activation of both Ca2+ and cAMP-dependent Cl- conductances in epididymal cells.
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PMID:Extracellular ATP activates both Ca(2+)- and cAMP-dependent Cl- conductances in rat epididymal cells. 856 54


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