How receptors talk to trimeric G proteins

Stimulated by hormones and sensory stimuli, serpentine receptors promote the release of GDP that is bound to the alpha subunit of trimeric G proteins and its replacement by GTP. Recent investigations have begun to define the sizes, shapes, and relative orientations of receptors and G proteins, the surfaces through which they interact with one another, and conformational changes in both sets of molecules that underlie receptor-catalyzed guanine-nucleotide exchange.     

Role of the prenyl group on the G protein gamma subunit in coupling trimeric G proteins to A1 adenosine receptors

The coupling of receptors to heterotrimeric G proteins is determined by interactions between the receptor and the G protein alpha subunits and by the composition of the betagamma dimers. To determine the role of the gamma subunit prenyl modification in this interaction, the CaaX motifs in the gamma1 and gamma2 subunits were altered to direct modification with different prenyl groups, recombinant betagamma dimers expressed in the baculovirus/Sf9 insect cell system, and the dimers purified. The activity of the betagamma dimers was compared in two assays: formation of the high affinity agonist binding conformation of the A1 adenosine receptor and receptor-catalyzed exchange of GDP for GTP on the alpha subunit. The beta1gamma1 dimer (modified with farnesyl) was significantly less effective than beta1gamma2 (modified with geranylgeranyl) in either assay. The beta1gamma1-S74L dimer (modified with geranylgeranyl) was nearly as effective as beta1gamma2 in either assay. The beta1gamma2-L71S dimer (modified with farnesyl) was significantly less active than beta1gamma2. Using 125I-labeled betagamma subunits, it was determined that native and altered betagamma dimers reconstituted equally well into Sf9 membranes containing A1 adenosine receptors. These data suggest that the prenyl group on the gamma subunit is an important determinant of the interaction between receptors and G protein gamma subunits.     

An aspartate residue in the extracellular loop of the N-methyl-D-aspartate receptor controls sensitivity to spermine and protons

To study the role of acidic residues in modulation of NMDA receptors by spermine, we used site-directed mutagenesis of receptor subunits and voltage-clamp recording in Xenopus oocytes. Sixteen glutamate and aspartate residues, located in the first two thirds of the putative extracellular loop of the NR1A subunit, were individually mutated. This region of NR1A shows homology with bacterial amino acid binding proteins, a bacterial polyamine binding protein, and a bacterial spermidine acetyltransferase. Mutation of D669 to asparagine (D669N), alanine (D669A), or glutamate (D669E) abolished the "glycine-independent" form of spermine stimulation in heteromeric NR1A/NR2B receptors. These mutations also markedly reduced inhibition by ifenprodil and by protons at NR1A/NR2B receptors. Mutations at the equivalent position (D690) in NR1B, which contains the insert encoded by exon 5, reduced the pH sensitivity of NR1B/NR2B receptors. Thus, the effects of mutations at D669 are not prevented by the presence of exon 5, and the influence of exon 5 is not prevented by mutations at D669 (D690 in NR1B). Mutations at NR1A (D669) had little or no effect on the potencies of glutamate and glycine and did not alter voltage-dependent block by Mg2+ or the "glycine-dependent" form of spermine stimulation. Surprisingly, the D669N and D669A mutations, but not the D669E mutation, reduced voltage-dependent block by spermine at NR1A/NR2 receptors. Mutations in NR2B at a position (D668) equivalent to D669 did not alter spermine stimulation or sensitivity to pH and ifenprodil. However, mutations D668N and D668A but not D668E in NR2B reduced voltage-dependent block by spermine. Screening of the negative charges at NR1A(D669) and NR2B(D668) may be involved in voltage-dependent block by spermine. D669 in NR1A could form part of a binding site for polyamines and ifenprodil and/or part of the proton sensor of the NMDA receptor. Alternatively, this residue may be critical for coupling of modulators such as spermine, protons, and ifenprodil to channel gating.     

Functional coupling of NKR-P1 receptors to various heterotrimeric G proteins in rat interleukin-2-activated natural killer cells

NKR-P1 molecules constitute a family of type II membrane receptors in natural killer (NK) cells that preferentially activate NK cell killing and release of interferon-gamma from these cells. Here, we demonstrate that anti-NKR-P1 enhances GTP binding in rat interleukin-2-activated NK cell membranes; GTP binding to Gi3alpha, Gsalpha, Gq,11alpha, and Gzalpha increased noticeably in these cell membranes after treatment with anti-NKR-P1. Western blot analysis of membrane proteins prepared from interleukin-2-activated NK cells reveals the presence of Gi1,2alpha, Gi3alpha, Goalpha, Gsalpha, Gq, 11alpha, Gzalpha, and G12alpha, but not G13alpha. However, only alphai3, alphas, alphaq,11, and alphaz, but not alphai1,2, alphao, alpha12, or alpha13 subunits when immunoprecipitated with the appropriate anti-G protein antibodies, are associated with NKR-P1 when immunoblotted with anti-NKR-P1. Reciprocally, NKR-P1 immunoprecipitated with anti-NKR-P1 is associated with alphai3, alphas, alphaq,11, and alphaz immunoblotted with anti-G proteins. These results are the first to demonstrate the physical and functional coupling of NKR-P1 to the heterotrimeric G proteins in NK cells.     

Reconstitution of receptors and GTP-binding regulatory proteins (G proteins) in Sf9 cells. A direct evaluation of selectivity in receptor.G protein coupling

The selectivity in coupling of various receptors to GTP-binding regulatory proteins (G proteins) was examined directly by a novel assay entailing the use of proteins overexpressed in Spodoptera frugiperda (Sf9) cells. Activation of G proteins was monitored in membranes prepared from Sf9 cells co-expressing selected pairs of receptors and G proteins (i.e. alpha, beta1, and gamma2 subunits). Membranes were incubated with [35S]guanosine 5'-(3-O-thio)triphosphate (GTPgammaS) +/- an agonist, and the amount of radiolabel bound to the alpha subunit was quantitated following immunoprecipitation. When expressed without receptor (but with beta1gamma2), the G protein subunits alphaz, alpha12, and alpha13 did not bind appreciable levels of [35S]GTPgammaS, consistent with a minimal level of GDP/[35S]GTPgammaS exchange. In contrast, the subunits alphas and alphaq bound measurable levels of the nucleotide. Co-expression of the 5-hydroxytryptamine1A (5-HT1A) receptor promoted binding of [35S]GTPgammaS to alphaz but not to alpha12, alpha13, or alphas. Binding to alphaz was enhanced by inclusion of serotonin in the assay. Agonist activation of both thrombin and neurokinin-1 receptors promoted a modest increase in [35S]GTPgammaS binding to alphaz and more robust increases in binding to alphaq, alpha12, and alpha13. Binding of [35S]GTPgammaS to alphas was strongly enhanced only by the activated beta1-adrenergic receptor. Our data identify interactions of receptors and G proteins directly, without resort to measurements of effector activity, confirm the coupling of the 5-HT1A receptor to Gz and extend the list of receptors that interact with this unique G protein to the receptors for thrombin and substance P, imply constitutive activity for the 5-HT1A receptor, and demonstrate for the first time that the cloned receptors for thrombin and substance P activate G12 and G13.     

Mutation of a highly conserved aspartate residue in the second transmembrane domain of the cannabinoid receptors, CB1 and CB2, disrupts G-protein coupling

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nM for hCRF, antagonized by the CRF antagonist alpha-helical CRF9-41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to [11-D-Thr,12-D-Phe]- and [13-D-His,14-D-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2alpha or -R2beta. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.     

Differential requirements of sodium for coupling of cannabinoid receptors to adenylyl cyclase in rat brain membranes

Sodium is generally required for optimal inhibition of adenylyl cyclase by Gi/o-coupled receptors. Cannabinoids bind to specific receptors that act like other members of the Gi/o-coupled receptor superfamily to inhibit adenylyl cyclase. However, assay of cannabinoid inhibition of adenylyl cyclase in rat cerebellar membranes revealed that concentrations of NaCl ranging from 0 to 150 mM had no effect on agonist inhibition. This lack of effect of sodium was not unique to cannabinoid receptors, because the same results were observed using baclofen as an agonist for GABAB receptors in cerebellar membranes. The lack of sodium dependence was region-specific, because assay of cannabinoid and opioid inhibition of adenylyl cyclase in striatum revealed an expected sodium dependence, with 50 mM NaCl providing maximal inhibition levels by both sets of agonists. This difference in sodium requirements between these two regions was maintained at the G protein level, because agonist-stimulated low Km GTPase activity was maximal at 50 mM NaCl in striatal membranes, but was maximal in the absence of NaCl in cerebellar membranes. Assay of [3H]WIN 55212-2 binding in cerebellar membranes revealed that the binding of this labeled agonist was sensitive to sodium and guanine nucleotides like other Gi/o-coupled receptors, because both NaCl and the nonhydrolyzable GTP analogue Gpp(NH)p significantly inhibited binding. These results suggest that differences in receptor-G protein coupling exist for cannabinoid receptors between these two brain regions.     

Pivotal role of leptin in insulin effects

The OB protein, also known as leptin, is secreted by adipose tissue, circulates in the blood, probably bound to a family of binding proteins, and acts on central neural networks regulating ingestive behavior and energy balance. The two forms of leptin receptors (long and short forms) have been identified in various peripheral tissues, a fact that makes them possible target sites for a direct action of leptin. It has been shown that the OB protein interferes with insulin secretion from pancreatic islets, reduces insulin-stimulated glucose transport in adipocytes, and increases glucose transport, glycogen synthesis and fatty acid oxidation in skeletal muscle. Under normoglycemic and normoinsulinemic conditions, leptin seems to shift the flux of metabolites from adipose tissue to skeletal muscle. This may function as a peripheral mechanism that helps control body weight and prevents obesity. Data that substantiate this hypothesis are presented in this review.     

Promiscuous coupling of receptors to Gq class alpha subunits and effector proteins in pancreatic and submandibular gland cells

Mice with deficiencies in one or more Gq class alpha subunit genes were used to examine the role of the alpha subunit in regulating Ca2+ signaling in pancreatic and submandibular gland cells. Western blot analysis showed that these cells express three of the four Gq class subunits, Galphaq, Galpha11, and Galpha14 but not Galpha15. Surprisingly, all parameters of Ca2+ signaling were identical in cells from wild type and four lines of mutant mice: 1) Galpha11-/-, 2) Galpha11-/-/Galpha14-/-, 3) Galpha14-/-/Galpha15-/-, and 4) Galphaq-/-/Galpha15-/-. These parameters included the Kapp for several Gq class coupled receptors, induction of [Ca2+]i oscillations by weak stimulation, and a biphasic [Ca2+]i response by strong stimulation. Furthermore, Ca2+ release from internal stores and Ca2+ entry were not affected in cells from any of the mutant mice. We conclude that Galphaq, Galpha11, and Galpha14 promiscuously couple several receptors (m3 muscarinic, bombesin, cholecystokinin, and alpha1 adrenergic) to effector proteins that activate both Ca2+ release from internal stores and Ca2+ entry.     

Multiple coupling of human D5 dopamine receptors to guanine nucleotide binding proteins Gs and Gz

We have demonstrated previously that D1 dopamine receptors are coupled to both Gs alpha and Go alpha. We examine here the coupling between human D5 dopamine receptors and G proteins in transfected rat pituitary GH4C1 cells. Similar to D1 receptors, cholera toxin treatment of cells reduced, but did not abolish, D5 agonist high-affinity binding sites, indicating D5 receptors couple to both Gs alpha and cholera toxin-insensitive G proteins. The interaction between D5 receptors and Gs alpha was confirmed by immunoprecipitation studies and by the ability of D5 receptors to stimulate adenylyl cyclase. Unlike D1 receptors, D5 receptors did not display any pertussis toxin-sensitive G-protein coupling to Go alpha or Gi alpha. D5 receptors were also not coupled to Gq alpha and were unable to mediate phosphatidylinositol metabolism. Instead, D5 sites appeared to be coupled to an AIF(-)4-sensitive, N-ethylmaleimide-resistant G protein. Anti-Gz alpha caused immunoprecipitation of 24.2 +/- 5.2% of G protein-associated D5 receptors, indicating coupling between D5 and Gz alpha. The coupling to Gz alpha was specific for D5 receptors, because similar associations were not detected between D1 receptors and Gz alpha.     

Selective interactions of mu-opioid receptors with pertussis toxin-sensitive G proteins: involvement of the third intracellular loop and the c-terminal tail in coupling

A cDNA encoding the rat mu-opioid receptor was expressed stably in a Rat-1 fibroblast cell line. Expression of this receptor was demonstrated with specific binding of the mu-opioid selective ligand [3H][D-Ala2,N-MePhe4,Gly5-ol]-enkephalin ([3H]DAMGO). In membranes of clone mu11 cells DAMGO produced a robust, concentration-dependent stimulation of basal high affinity GTPase activity. Cholera toxin-catalyzed [32P]ADP-ribosylation in membranes of this clone labelled a 40 kDa Gi family polypeptide(s) that was markedly enhanced by the addition of DAMGO. Antisera against Gi2alpha and Gi3alpha were both able to immunoprecipitate a [32P]-radiolabelled 40 kDa polypeptide(s) from DAMGO and cholera-toxin treated membranes of clone mu11, indicating that the mu-opioid receptor was able to interact effectively with both Gi2 and Gi3 in Rat-1 fibroblasts. A series of peptides derived from the delta-opioid receptor sequence were assessed for their ability to modify agonist-stimulated G protein activation and [3H] agonist binding to the receptor. In membranes from the clone mu11, specific binding of [3H]DAMGO was reduced by peptides corresponding to the NH2-terminal region of the third intracellular loop (i3.1) and the carboxyl-terminal tail (i4) of this receptor. Agonist stimulated GTPase activity and DAMGO dependent cholera toxin-catalyzed [32P]ADP-ribosylation were inhibited by peptides derived from the proximal (i3.1) and the distal portion (i3.3) of the third intracellular loop. Peptide i3.1 also inhibited DAMGO-stimulated [35S]guanosine-5'-O-(3-thio)triphosphate ([35S]GTP-gammaS) binding in the same membranes. In contrast, peptides derived from the second intracellular loop were without any effect.     

A new class of phosphotransferases phosphorylated on an aspartate residue in an amino-terminal DXDX(T/V) motif

When incubated with their substrates, human phosphomannomutase and L-3-phosphoserine phosphatase are known to form phosphoenzymes with chemical characteristics of an acyl-phosphate. The phosphorylated residue in phosphomannomutase has now been identified by mass spectrometry after reduction of the phosphoenzyme with tritiated borohydride and trypsin digestion. It is the first aspartate in a conserved DVDGT motif. Replacement of either aspartate of this motif by asparagine or glutamate resulted in complete inactivation of the enzyme. The same mutations performed in the DXDST motif of L-3-phosphoserine phosphatase also resulted in complete inactivation of the enzyme, except for the replacement of the second aspartate by glutamate, which reduced the activity by only about 40%. This suggests that the first aspartate of the motif is also the phosphorylated residue in L-3-phosphoserine phosphatase. Data banks contained seven other phosphomutases or phosphatases sharing a similar, totally conserved DXDX(T/V) motif at their amino terminus. One of these (beta-phosphoglucomutase) is shown to form a phosphoenzyme with the characteristics of an acyl-phosphate. In conclusion, phosphomannomutase and L-3-phosphoserine phosphatase belong to a new phosphotransferase family with an amino-terminal DXDX(T/V) motif that serves as an intermediate phosphoryl acceptor.     

Differential coupling of mu-, delta-, and kappa-opioid receptors to G alpha16-mediated stimulation of phospholipase C

The mu-opioid receptor has recently been shown to stimulate phosphoinositide-specific phospholipase C via the pertussis toxin-sensitive G16 protein. Given the promiscuous nature of G16 and the high degree of resemblance of signaling properties of the three opioid receptors, both delta- and kappa-opioid receptors are likely to activate G16. Interactions of delta- and kappa-opioid receptors with G16 were examined by coexpressing the opioid receptors and G alpha16 in COS-7 cells. The delta-selective agonist [D-Pen2,D-Pen5] enkephalin potently stimulated the formation of inositol phosphates in cells coexpressing the delta-opioid receptor and G alpha16. The delta-opioid receptor-mediated stimulation of phospholipase C was absolutely dependent on the coexpression of G alpha16 and exhibited appropriate ligand selectivity and dose dependency. Similar transfection studies revealed only weak stimulation by the mu-opioid receptor, whereas the kappa-opioid receptor produced moderate phospholipase C activity. G alpha16 thus appeared to interact differentially with the three opioid receptors. Radioligand binding assays indicate that the mu-opioid receptor was expressed at a lower level than those of the delta- and kappa-opioid receptors. To examine if differential coupling to G alpha16 is prevalent, a panel of Gs- or Gi-coupled receptors was coexpressed with G alpha16 in COS-7 cells and assayed for agonist-induced stimulation of phospholipase C. Activation of alpha2- and beta2-adrenergic, dopamine D1 and D2, adenosine A1, somatostatin-1 and -2, C5a, formyl peptide, and luteinizing hormone receptors all resulted in stimulation of phospholipase C, with maximal stimulations ranging from 1.5- to almost 17-fold. These findings suggest that the promiscuous G alpha16 can in fact discriminate among different receptors and that such preferential interaction might in part be due to the abundance of receptors.     

Coronary arteriolar dilation to acidosis: role of ATP-sensitive potassium channels and pertussis toxin-sensitive G proteins

BACKGROUND: We previously demonstrated that coronary arteriolar dilation in response to acidosis is mediated by the opening of ATP-sensitive potassium (KATP) channels. However, the signal transduction involved in the KATP-channel activation during acidosis has not been elucidated. A recent study in cardiac myocytes implied that pertussis toxin (PTX)-sensitive G proteins may be involved in the signal transduction for KATP-channel activation. However, it remains unclear whether this transduction process also occurs in the vascular tissue and, in particular, whether it exerts functional dilation in response to acidosis. METHODS AND RESULTS: To examine the signaling pathway for acidosis-induced dilation, porcine coronary arterioles were isolated, cannulated, and pressurized for in vitro study. The GTPase activity in reconstituted G proteins was examined at different levels of pH. Extravascular acidosis (pH 7.3 to 7.0) produced a graded dilation of coronary arterioles. This dilation was not affected by removal of endothelium but was significantly attenuated after inhibition of KATP channels and G proteins by glibenclamide and PTX, respectively. Glibenclamide and PTX attenuated the acidosis-induced arteriolar dilation to the same extent, and combined administration of both inhibitors did not further inhibit the vasodilation. These results indicated that both inhibitors act on the same vasodilatory pathway. Furthermore, vasodilation of coronary arterioles to the KATP-channel opener pinacidil and to the endothelium-independent vasodilator sodium nitroprusside was not affected by PTX. Because PTX inhibited acidosis-induced vasodilation without inhibiting KATP-channel function, it is suggested that PTX inhibits the vasodilatory pathway upstream from KATP channels. GTPase activity in reconstituted G proteins was significantly enhanced by a reduction in pH, indicating that G proteins were directly activated by acidosis. CONCLUSIONS: On the basis of these findings, we conclude that acidosis-induced coronary arteriolar dilation is mediated by the opening of smooth muscle KATP channels through the activation of PTX-sensitive G proteins.     

Pivotal role of mitochondrial calcium uptake in neural cell apoptosis and necrosis

Perturbed cellular calcium homeostasis has been implicated in both apoptosis and necrosis, but the role of altered mitochondrial calcium handling in the cell death process is unclear. The temporal ordering of changes in cytoplasmic ([Ca2+]C) and intramitochondrial ([Ca2+]M) calcium levels in relation to mitochondrial reactive oxygen species (ROS) accumulation and membrane depolarization (MD) was examined in cultured neural cells exposed to either an apoptotic (staurosporine; STS) or a necrotic (the toxic aldehyde 4-hydroxynonenal; HNE) insult. STS and HNE each induced an early increase of [Ca2+]C followed by delayed increase of [Ca2+]M. Overexpression of Bcl-2 blocked the elevation of [Ca2+]M and the MD in cells exposed to STS but not in cells exposed to HNE. The cytoplasmic calcium chelator BAPTA-AM and the inhibitor of mitochondrial calcium uptake ruthenium red prevented both apoptosis and necrosis. STS and HNE each induced mitochondrial ROS accumulation and MD, which followed the increase of [Ca2+]M. Cyclosporin A prevented both apoptosis and necrosis, indicating critical roles for MD in both forms of cell death. Caspase activation occurred only in cells undergoing apoptosis and preceded increased [Ca2+]M. Collectively, these findings suggest that mitochondrial calcium overload is a critical event in both apoptotic and necrotic cell death.     

The role of G-regulatory proteins in the intracellular mechanism of cardiac angiotensin-II receptors

High-affinity angiotensin II receptors have been identified in cardiac tissue of many animal species. In the heart, angiotensin II exerts positive inotropic and chronotropic effects, constricts coronary vessels, and stimulates cell growth. In vascular smooth muscle and adrenal cortex angiotensin II interacts with guanidine nucleotide regulatory proteins because GTP-gamma-S causes dissociation of the radioligand from its receptor. To investigate whether angiotensin II interacts with guanidine nucleotide regulatory proteins also in cardiac tissue, we studied the effects of GTP-gamma-S on [Sar1, Ile8]-angiotensin II binding to angiotensin II receptor subtypes (AT1 and AT2) in hearts obtained from 16- to 20-week-old Sprague-Dawley rats. We employed an in situ technique performed on frozen tissue sections. Competition experiments performed with the nonpeptide inhibitors losartan and PD123177 allowed identification of both AT1 and AT2 angiotensin II receptors in rat heart. These receptors were present in comparable amounts. In a different set of experiments the effects of GTP-gamma-S (100 microM) on radioligand displacement from AT1 and AT2 receptors were studied. GTP-gamma-S caused a progressive dissociation of the radioligand from the AT1 receptor indicating that this receptor interacts with guanidine nucleotide regulatory proteins. In contrast, the AT2 receptor does not appear to directly interact with guanidine nucleotide regulatory proteins. In summary, the study shows that both angiotensin II receptor subtypes are present in rat heart and that guanidine nucleotide regulatory proteins are implicated in the signal transduction mechanism of the cardiac AT1 receptor.     

G proteins and hypertension: an alternative candidate gene approach

Hypertension affects approximately 20 to 30% of individuals in industrialized countries, and is commonly believed to develop on the basis of both genetic and environmental factors. The identification of genes susceptible to the most frequent form of hypertension, commonly referred to as "essential" hypertension, is hampered by the fact that blood pressure is a poorly defined phenotype that is modulated by multiple factors, such as gender, race, body mass etc., and that the definition of hypertension depends on a rather arbitrarily chosen cut-off value. Hence, more progress has been made in the identification of genes responsible for rare autosomal dominant forms of hypertension, such as Liddle's disease. This review focuses on an experimental approach that attempts to define candidate genes for essential hypertension using immortalized cells from well characterized normotensive and hypertensive subjects. From the presently available results, one attractive speculation is that an increased intracellular signal transduction caused by an enhanced reactivity of Gj-type G proteins represents a genetically fixed trait that renders affected individuals susceptible to essential hypertension.     

NMDA receptors play an anti-aggregating role in human platelets

We Describe a case of ovarian serous cystadenoma having Sertoli-Leydig cell tumor, well differentiated, in the cystic septum. Well differentiated Sertoli-Leydig cell tumor coexisting with other tumor, including serous tumor, has not yet been described. In all cases of Sertoli-Leydig cell tumor with heterologous components or other tumors, the androblastomatous components are intermediately or poorly differentiated. The present case revealed a well differentiated Sertoli-Leydig cell tumor arising in a septum of serous cystadenoma, as a circumscribed nodule. With these findings, we discuss the possibility of this Sertoli-Leydig cell tumor, considered a mural nodule, which is well established in cystic common epithelial tumors of the ovary.     

The role of central muscarinic and nicotinic receptors in the regulation of sodium and potassium renal excretion

The effect of intraseptal injection of carbachol and nicotine on urinary output of Na+ and K+ in untreated rats as well as in animals pretreated with locally injected atropine, hexamethonium, dibenamine and propranolol was studied in order to evaluate the relative role played by central muscarinic and nicotinic receptors in the regulation of salt and water renal excretion. The injection of 30-250 nmol of nicotine into the medial septal area caused a dose-dependent increase in Na+ and K+ urinary output whereas urine volume was little affected. The effect of 30 nmol of nicotine was blocked by pretreatment with 100 nmol of hexamethonium. In addition, pretreatment with 5 nmol of either hexamethonium or atropine partially antagonized the natriuretic and kaliuretic effect of 1 nmol of carbachol. Also the alpha-blocking agent, dibenamine (150 nmol) antagonized, while the beta-blocker, propranolol (100 nmol) significantly enhanced the effect of carbachol. Propranolol (100 nmol) alone caused a small, but significant increase in Na+ and K+ renal excretion. These results indicate that stimulation of both muscarinic and nicotinic receptors in the septal area, as caused by carbachol, elicits increased disposition of Na+ and K+ by the kidneys. Also, part of the effects of carbachol appear to be mediated by the release of endogenous catecholamines, acting on central alpha receptors to increase Na+ and K+ urinary excretion. On the other hand, simultaneous activation of beta-receptors by the released amines would partially inhibit this effect.