Beta-cyclopentamethylenepropionic Acid Research Articles

Fluorescence study of neurohypophyseal hormones and their analogues. Distance distributions in a series of arginine-vasopressin analogues.

Analogues of arginine-vasopressin (AVP) in which substitution of the proline residue in position 7 (by either sarcosine or N-methylalanine) combined with replacement of the cysteine residue in position 1 were the subject of a fluorescence and molecular mechanics study. We obtained two groups of analogues: selective antidiuretic agonists (cysteine or beta-mercaptopropionic acid in position 1) and pressor and uterotonic antagonists (deaminopenicillamine or beta-mercapto-beta, beta-cyclopentamethylenepropionic acid in position 1). Using frequency-domain measurements of fluorescence resonance energy transfer (FRET) we estimated the distance distribution between the phenolic ring of Tyr2 and the disulphide bridge Cys1-Cys6. We also analyzed acrylamide quenching of tyrosyl fluorescence to determine the exposure of the tyrosyl ring to the solvent. Results from fluorescence experiments were compared with those from Monte Carlo simulation (ECEPP/3 force-field).

Arginine vasopressin mediates the chloroquine induced increase in renal sodium excretion

We postulated that chloroquine increases plasma arginine vasopressin (AVP) concentrations thus altering renal Na+ clearance. Therefore, we studied a relationship between plasma AVP concentrations and urinary Na+ output in separate groups of Sprague-Dawley (SD) rats administered chloroquine (3 micrograms/min) for 1 h 20 min. We also monitored Na+ excretion rates in Brattleboro AVP-deficient Di rats challenged with hypotonic saline load and administered chloroquine for 1 h 20 min. To establish whether chloroquine-induced changes in renal Na+ excretion were mediated via AVP V1 receptors, we studied Na+ excretion rates in groups of SD rats administered chloroquine or AVP in the presence of AVP V1 receptor antagonist (1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid)-2-O-methyltyrosine arginine vasopressin (d(CH2)5(Tyr(Me)2) AVP) at 11 pmol/min for 1 h 20 min. The Na+ excretion rate rose significantly (P < 0.01) from a pretreatment level of 9.8 +/- 1.0 mumol/min to a peak of 14.1 +/- 0.9 mumol/min in SD rats (n = 7) administered chloroquine. The Na+ excretion rate remained unaltered around 8.5 mumol/min in rats simultaneously administered chloroquine and the AVP V1 receptor antagonist. This compared with control rats (8.1 +/- 0.5 mumol/min, n = 7) and animals administered AVP V1 receptor antagonist alone (8.7 +/- 0.6 mumol/min, n = 7). Chloroquine did not affect urine flow, Na+ or K+ excretion rates in Brattleboro AVP-deficient Di rats. Administration of AVP alone was associated with significant increases in renal Na+ excretion rate. Blockade of AVP V1 receptors abolished the AVP-dependent increase in urinary Na+ loss. We conclude that at least part of the chloroquine-induced increase in Na+ excretion is mediated by chloroquine stimulating an increase in plasma AVP concentration.

Modulation of the effect of arginine-vasopressin on water and ion transport in the newt early distal tubule and frog urinary bladder by V1-antagonists.

In the early distal tubule of the newt Triturus vulgaris L., 1 nM arginine-vasopressin (AVP) increased water reabsorption; the fractional reabsorption of Na+ was elevated from 46.2 +/- 6.9% to 67.8 +/- 3.9% (P < 0.001), of Cl- from 52.7 +/- 6.7% to 73.1 +/- 3.5% (P < 0.001), of Mg2+ from 48.0 +/- 7.7% to 71.7 +/- 6.3% (P < 0.001). When V1-receptors were blocked by 1 nM peptide V1-antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(O-methyl) Tyr]-[Arg8]vasopressin, 1 nM AVP increased the fractional reabsorption of fluid by 8.9% of Na+ by 10.7% and of Cl- by 11.2%, as compared with the effect of AVP alone. The fractional reabsorption of Ca2+ after addition of AVP did not differ from control; when V1-receptors were blocked in the presence of AVP, the fractional reabsorption of Ca2+ was increased by AVP. The V1-receptor block in the presence of AVP did not change the fractional reabsorption of Mg2+. Experiments on the urinary bladder of the frog Rana temporaria L. showed that 1 nM SR 49059, a non-peptide antagonist of V1a-receptors, like the peptide V1-antagonist, enhanced the AVP effect by 29%. Inhibition of protein kinase C activity by calphostin C (1 nM) mimicked the effect of V1-antagonists; the AVP hydroosmotic effect was increased by 60%. The results obtained indicate that V1-receptors modulate the effects of V2-receptor activation: their block is accompanied by an enhancement of the AVP hydroosmotic effect in the frog urinary bladder and by an increase of Na+ and Cl- reabsorption in the newt early distal tubule. The enhancement of the AVP effect owing to the V1-receptor activation seems to be mediated by a decrease in protein kinase C activity.

Vasopressin actions on area postrema neurons in vitro.

The area postrema (AP) is a circumventricular organ located on the dorsal surface of the medulla. Substantial evidence suggests that the AP is an important site involved in cardiovascular regulation. Arginine vasopressin (AVP) is thought to act at the AP to increase the sensitivity of the baroreceptor reflex. We have therefore examined the effects of AVP on AP neurons with the use of extracellular single unit recordings in vitro. Coronal medullary brain slices (thickness = 400 microns) were obtained from male Sprague-Dawley rats and maintained in oxygenated artificial cerebrospinal fluid (aCSF). The slices were perfused with AVP (10(-8) to 10(-6) M), and the effect on single AP neurons was recorded. A total of 79 AP neurons was tested of which 50 (63.3%) were excited by AVP and 5 (6.3%) were inhibited, whereas the remaining 24 (30.3%) cells were unaffected. The excitatory effects of AVP were dose dependent: firing rate increased 92.6 +/- 25.8% at 10(-8) M, 289.4 +/- 53.9% at 10(-7) M, and 456.8 +/- 113.1% at 10(-6) M, respectively. We also examined whether these effects of AVP resulted from direct actions of this peptide on AP cells by testing if responses were retained during blockade of synaptic transmission (achieved by perfusion with a low Ca(2+)-high Mg2+ aCSF) in 11 cells excited by AVP. Nine of these cells were excited by AVP during such synaptic blockade. Finally, we demonstrated that the excitatory responses of five AP cells to AVP were all totally abolished by perfusion of slices with aCSF containing the V1 antagonist ([1-beta-mercapto-beta,beta-cyclopentamethylene propionic acid,2-(O-methyl)tyrosine]-Arg8-vasopressin; Peninsula Laboratories, 10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)

A new tritiated oxytocin receptor radioligand--synthesis and application for localization of central oxytocin receptors.

A new tritiated oxytocin antagonist radioligand was synthesized by introducing a tritiated propionic acid residue into the free amino group of ornithine in position 8 of the parent peptide [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(O-methyl)-tyrosine, 4-threonine, 8-ornithine, 9-tyrosylamide]vasotocin (OTA), that was previously described. The tritiated compound [3H][1-(beta-mercapto-beta,beta-cyclo-pentamethylene propionic acid), 2-(O-methyl)-tyrosine, 4-threonine, 8-(N6-propionyl)-ornithine, 9-tyrosylamide]vasotocin ([3H]PrOTA) was obtained in good yield with high specific activity (100 Ci/mmol). [3H]PrOTA exhibited the same affinity (Kd = 0.8 nM) and selectivity for the myometrial oxytocin receptor as the iodinated antagonist [125I]OTA. Autoradiographic localization of oxytocin receptors in the rat brain showed specific binding sites for [3H]PrOTA within regions of the limbic system, the neocortex, and hypothalamus, which is consistent with the binding pattern obtained with [125I]OTA. The high specific activity in combination with the long half-life of tritium and its low radiotoxicity as compared to iodine-125 makes the new tritiated antagonist a valuable tool for pharmacological studies.

Angiotensin mediates stimulation of ventilation after vasopressin V1 receptor blockade

We tested the hypothesis that respiration would be stimulated after vasopressin (AVP) V1 receptor blockade because of disinhibition and activation of the renin-angiotensin system. Intravenous infusion of angiotensin II (ANG II) stimulates respiration, presumably centrally, via circumventricular organs. In the present study, the AVP V1 receptor antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid),2-(O-methyl)tyrosine]-Arg8-AVP (PMP; 10 micrograms/kg i.v.) was administered to six awake resting dogs. Measurements were made 30 min prior, and 60 min subsequent, to injection of PMP (protocol 1). In three other protocols, the ANG II blocker saralasin (0.5 microgram.kg-1.min-1 i.v.) was infused starting 20 min before PMP (protocol 2) and 30 min after PMP (protocol 4) and saline was infused (0.2 ml/min) over 90 min as a control (protocol 3). After PMP in protocol 1, alveolar ventilation increased and arterial PCO2 decreased (approximately 3 Torr). ANG II receptor blockade prevented (protocol 2) and reversed (protocol 4) respiratory stimulation by PMP. Despite ventilatory stimulation, plasma renin activity and ANG II were not increased after PMP relative to control (protocol 3). We conclude that AVP acts at V1 receptors to inhibit formation of brain ANG II. Brain ANG II must modulate respiratory control via a circumventricular organ, because systemically administered saralasin, which does not cross the blood-brain barrier, blocked stimulation of respiration.

Role of septal vasopressin innervation in paternal behavior in prairie voles (Microtus ochrogaster).

After being paired with females, male prairie voles show major changes in their social behaviors among which is an increase in paternal responsiveness. These changes are accompanied by fluctuations in the density of the [Arg8]vasopressin-immunoreactive (AVP-ir) fibers in the lateral septum, suggesting that septal AVP might be involved in these changes. To explore a possible involvement of septal AVP in paternal responsiveness, we tested whether injections of saline, AVP, or the V1a receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid),2-(O-methyltyrosine]AVP [d(CH2)5Tyr(Me)AVP] into the lateral septum influenced the four most prominent paternal activities displayed by male prairie voles; grooming, crouching over, contacting, and retrieving pups. In a first experiment, sexually inexperienced males received a single injection of AVP, saline, or d(CH2)5Tyr(Me)AVP in the lateral septum, after which their paternal responsiveness was recorded during a 10-min period. AVP-injected animals spent more time contacting and crouching over pups, while d(CH2)5Tyr(Me)AVP-injected animals spent less time grooming pups than saline-injected animals. In a follow-up study, one group of animals received an injection of AVP preceded by an injection of saline or d(CH2)5Tyr(Me)-AVP into the lateral septum. A second group of animals received an injection of saline preceded by an injection of saline or d(CH2)5Tyr(Me)AVP into the lateral septum. In both groups, animals spent less time grooming, crouching over, and contacting pups if they had first been injected with d(CH2)5Tyr(Me)AVP. Control experiments suggested that the effects of AVP on paternal responsiveness were dose- and site-specific. These data suggest that septal AVP enhances paternal responsiveness by a V1a receptor-mediated mechanism.

Demonstration of oxytocin receptors in porcine corpora lutea: effects of the cycle stage and the distribution on small and large luteal cells.

Recent investigations have demonstrated an inhibitory effect of oxytocin (OXT) on luteal cell progesterone (P) release under in vitro conditions. This inhibitory effect was counteracted by an OXT antagonist, indicating that it was receptor-mediated. In the present investigation, we demonstrated the presence of OXT binding sites in porcine luteal tissue using a radioiodinated OXT antagonist, [1-(beta mercapto-beta,beta-cyclopentamethylene propionic acid),2-(ortho-methyl)-Tyr2-Thr4-Orn8-Tyr-NH2] vasotocin (OTA), as ligand. For membrane fractions of porcine luteal tissue, Kd values of 0.7-0.8 nM were obtained; these are comparable to those of porcine myometrial fractions, measured under the same experimental conditions. Competition studies with luteal membrane fractions yielded a Ki(OXT) of 10(-9) M. This is a dose of OXT that exerts inhibitory effects on P release under both in vitro and in vivo conditions. To evaluate putative variations of luteal OXT receptor concentrations during the estrous cycle, membrane fractions prepared from corpora lutea (CL) of the early or midluteal (Days 2-6) and late luteal phase (Days 9-11) were used. While no differences in Kd values were observed, OXT binding capacities were significantly (p < 0.05) higher in CL from the early/midluteal phase (Bmax(E/M) = 116 +/- 12 fmol/mg protein) compared to CL from the late luteal phase (Bmax(L) = 65 +/- 10 fmol/mg protein). OXT binding sites were present in both small (SLC) and large luteal cells (LLC). SLC but not LLC responded to hCG with a significant increase of OXT binding sites, whereas E2 augmented OXT receptor binding in SLC as well as in LLC.(ABSTRACT TRUNCATED AT 250 WORDS)

Secretion of immunoreactive endothelin-1 by capillary and microvascular endothelium of human brain.

Modulation of immunoreactive endothelin-1 (IR-ET-1) production by vasoactive substances was investigated in cultured endothelial cells (EC) derived from capillaries and microvessels of human brain. Peptides, catecholamines, thrombin, protein kinase C-activating phorbol ester, and calcium ionophore enhanced the secretion of IR-ET-1. The known vasoconstrictive peptides, angiotensin II (Ang II) and arginine-vasopressin (AVP) dose-dependently stimulated the endothelial secretion of IR-ET-1. The angiotensin and vasopressin-inducible production of IR-ET-1 was completely inhibited by their respective receptor antagonists [Sar1, Ala8]-angiotensin II and [1-6 (beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-O-methyl-tyrosine]. The results indicate that the peptide-stimulated secretion of IR-ET-1 is receptor-mediated in EC which have specific angiotensin II and arginine-vasopressin receptors. These findings represent the first demonstration of IR-ET-1 production by capillary and microvascular endothelium of human brain.

Central effects of endothelin-1 on vasopressin release, blood pressure, and renal solute excretion.

To assess central effects of endothelin-1 (ET-1) on plasma arginine vasopressin (AVP), plasma atrial natriuretic peptide (ANP), blood pressure, heart rate, and renal solute excretion, ET-1 dissolved in the artificial cerebrospinal fluid was infused intracerebroventricularly (icv) at a dose of either 0.35 ng.kg-1.min-1 (0.14 pmol; LET) or 3.5 ng.kg-1.min-1 (1.4 pmol; HET) for 45 min in conscious rats. In the control study, ET-1 was omitted from the vehicle. Moreover, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid),2-O-methyl-tyrosine]AVP (TMeAVP), a V1-blocker, and prazosin, an alpha 1-blocker, were peripherally administered. In the LET group, mean arterial blood pressure (MABP) increased without any changes in heart rate, plasma AVP and ANP, and renal solute excretion. In the HET group, MABP markedly increased with rises in plasma AVP and ANP, but urine flow, urinary osmolality, and urinary Na and K excretion decreased. TMeAVP attenuated the pressor response to ET-1, abolished rises in plasma ANP, and partially restored falls in urine flow. Prazosin after TMeAVP obviated the pressor response to ET-1. In the control, these parameters did not change, except for an increase in urinary solute excretion. These results showed that icv ET-1 stimulates the sympathetic nervous activity and plasma AVP, leading to increased blood pressure and renal vasoconstriction, with a reduction in renal water and electrolyte excretion.

Molecular size characterization of oxytocin receptors in rabbit amnion.

The binding of oxytocin (OT) to receptors in rabbit amnion cells stimulates PGE2 release. We previously studied the binding characteristics, changes in receptor concentration during pregnancy, and agonist specificity of OT action on amnion cells. In this study the molecular size of OT receptors in rabbit amnion was estimated by photoaffinity labeling, radiation inactivation, and gel filtration of solubilized receptor, using an iodinated OT antagonist, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid, 2-O-methyltyrosine,4-threonine,8-ornithine,9-tyrosylamide]vasotoci n (OTA), or [3H]OT. Two electrophoretic bands, about 50 and 65 kilodaltons (kDa), were specifically covalently labeled with azidobenzoyl-[125I]OTA. Both sizes correspond to that determined by radiation inactivation, about 55 kDa, using [3H]OT binding to assess the fraction of receptor sites remaining. When we used [125I]OTA, the radiation inactivation size was about 30 kDa. These differences in radiation inactivation size suggest that the receptor binding site is comprised of more than one domain and that the binding of the antagonist involves fewer points of interaction than does OT. The molecular size of the receptor estimated from [125I]OTA binding by detergent-solubilized extracts of amnion membranes was about 350 kDa, as determined by gel filtration on columns of Sepharose 6B. Although the functional size of the receptor is about 65 kDa, it appears to be closely associated with other membrane proteins. The size estimates of amnion OT receptors agree with those in rabbit myometrium and rat mammary gland, both of which differ from amnion by contracting in response to OT. Despite different responses, OT receptors in different tissues appear to be very similar in size.

Molecular size characterization of oxytocin receptors in rabbit amnion

The binding of oxytocin (OT) to receptors in rabbit amnion cells stimulates PGE2 release. We previously studied the binding characteristics, changes in receptor concentration during pregnancy, and agonist specificity of OT action on amnion cells. In this study the molecular size of OT receptors in rabbit amnion was estimated by photoaffinity labeling, radiation inactivation, and gel filtration of solubilized receptor, using an iodinated OT antagonist, [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid, 2-O-methyltyrosine,4-threonine,8-ornithine,9-tyrosylamide]vasotoci n (OTA), or [3H]OT. Two electrophoretic bands, about 50 and 65 kilodaltons (kDa), were specifically covalently labeled with azidobenzoyl-[125I]OTA. Both sizes correspond to that determined by radiation inactivation, about 55 kDa, using [3H]OT binding to assess the fraction of receptor sites remaining. When we used [125I]OTA, the radiation inactivation size was about 30 kDa. These differences in radiation inactivation size suggest that the receptor binding site is comprised of more than one domain and that the binding of the antagonist involves fewer points of interaction than does OT. The molecular size of the receptor estimated from [125I]OTA binding by detergent-solubilized extracts of amnion membranes was about 350 kDa, as determined by gel filtration on columns of Sepharose 6B. Although the functional size of the receptor is about 65 kDa, it appears to be closely associated with other membrane proteins. The size estimates of amnion OT receptors agree with those in rabbit myometrium and rat mammary gland, both of which differ from amnion by contracting in response to OT. Despite different responses, OT receptors in different tissues appear to be very similar in size.

Vasopressin-induced antipyresis in the medial amygdaloid nucleus of conscious rats.

Experiments were undertaken to characterize a possible receptor mediating antipyretic action of arginine vasopressin (AVP) within the medial amygdaloid nucleus (meA) in the conscious rat. Additional experiments were directed at determining whether the action of endogenously released AVP can be revealed in the meA during fever in the conscious rat. These objectives were achieved using vasopressin analogues directed against vasopressor (V1a) and antidiuretic (V2) receptors. Bilateral injection of AVP (40 pmol) into the meA of conscious rats suppressed fever evoked by intracerebroventricular (icv) administration of prostaglandin E1 (PGE1, 50 ng). The V2 receptor agonist 1-desamino-8-D-AVP (40 pmol) injected into the meA evoked only moderate antipyresis compared with AVP, possibly because of interaction of this agonist with V1a receptors. The antipyretic effect of AVP was blocked when injection of the peptide was preceded by a bilateral injection of the V1a antagonist 1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-2-(O-methyl)tyrosine AVP [d(CH2)5Tyr(Me)AVP, 400 pmol] into the meA. Injection of d(CH2)5Tyr(Me)AVP alone into the meA was without significant effect on afebrile core temperature. Injection of d(CH2)5Tyr(Me)AVP or 1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid)-2-D-valine,4-valine AVP (a V2 antagonist) alone into the meA before icv PGE1 resulted in fevers that were not significantly different from artificial cerebrospinal fluid controls. These data are consistent with the possibility that AVP might act within the meA to evoke antipyresis via receptors that resemble V1a (vasopressor) receptors. However, the action of AVP endogenously released into the meA does not appear to be an absolute requisite in the normal modulation of PGE1 fever.

Pharmacological characterization of V1a vasopressin receptors in the rat cortical collecting duct.

Vasopressin receptors in distal segments of the rat nephron were identified in isolated tubules using two labeled ligands: the [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-(O-methyl)tyrosine,4-threonine,8-ornithine,9-125I-tyrosylamide]- vasotocin [125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]OVT] and the linear analogue, Phaa1,D-Tyr(Me)2,Phe3,Gln4,Asn5,Arg6, Pro7,Arg8,125I-Tyr-NH2(9) [125I-Tyr-NH2(9)-linear antagonist (LA)-V1a)]. Specific 125I-d(CH2)5[Tyr(Me)2,Thr4,Tyr-NH2(9)]-OVT binding to cortical collecting ducts (CCD) was saturable with incubation time and dose, reversible after elimination of free ligand, and characterized by the following rank order for recognition of vasopressin analogues: desGly9-d-(CH2)5-[Tyr(Et)2,Val4]arginine vasopressin (AVP) greater than or equal to d(CH2)5[Tyr-(ET)2,Val4]AVP greater than or equal to AVP greater than or equal to d(CH2)5[Tyr(Me)2]AVP = 1-desamino-8-D-arginine vasopressin (DDAVP) greater than or equal to Tyr-NH2(9)-LA-V1a greater than [8-arginine]vasotocin (AVT) greater than d(CH2)5[Tyr(Me)2, Thr4,Tyr-NH2(9)]OVT greater than oxytocin (OT) greater than [Phe2,Orn8]VT much greater than [Thr4,Gly7]-OT. Scatchard plots of dose-dependent 125I-Tyr-NH2(9)-LA-V1a binding to medullary thick ascending limbs (MTAL), CCD, and outer medullary collecting ducts (OMCD) revealed the presence of high- and low-affinity binding sites corresponding to V1a and V2 vasopressin receptors, respectively; the densities of V1a receptors are approximately 20% of the total number of vasopressin receptors in CCD and 5% in MTAL and OMCD.

A vasopressin analog that binds but does not activate V1 or V2 vasopressin receptors is not internalized into cells that express V1 or V2 receptors.

To assess whether receptor binding is sufficient to initiate vasopressin receptor endocytosis in cells expressing the vasopressin V1 or V2 receptors, we synthesized a novel fluorescent-labeled vasopressin analog, [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(O-ethyl)-D-tyrosine, 4-valine, 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-CLVP), that binds to vasopressin receptors but does not activate intracellular events such as the mobilization of intracellular calcium or the activation of adenylate cyclase. We compared the manner in which this analog was endocytosed in cells expressing V1 (A-10, rat smooth muscle cells) or V2 (LLC-PK1, porcine kidney cells) receptors with that of a full agonist, [1-(beta-mercaptopropionic acid), 8-lysine-N6-carboxytetramethylrhodamine] vasopressin (R-MLVP) [Lutz et al. (1990) J. Biol. Chem. 265, 4657-4663; Lutz et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87,6507-6511]. We showed that R-CLVP bound to both types of receptors with good affinity. It failed to increase cyclic AMP concentrations in LLC-PK1 cells and did not increase the mobilization of intracellular calcium in A-10 cells. It bound to the surface of both these cell types in a diffuse manner and it did not undergo receptor endocytosis in either cell type. In contrast, R-MLVP, an agonist that bound to both receptor subtypes and elicited changes in intracellular cyclic AMP and calcium, bound to the surface of these cells in a diffuse manner at early times after exposure, and rapidly underwent endocytosis. We conclude that binding of vasopressin to its receptors alone is insufficient to cause receptor endocytosis, and other events distal to the receptor are required to initiate endocytosis. R-CLVP should be a useful analog in determining the factors responsible for initiating receptor endocytosis.

Effects of amiloride analogues on AVP binding and activation of V1-receptor-expressing cells

We tested the interactions between amiloride analogues and V1 vascular arginine vasopressin (AVP) receptors of human platelets, rat glomerular mesangial cells, and A7r5 smooth muscle cells by using radioligand binding techniques, intracellular calcium monitoring, platelet aggregation, and cell contraction techniques. Amiloride analogues were competitive inhibitors of both the agonist [3H]AVP and the tritiated V1 antagonist [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyl)tyrosine]AVP ([3H]d(CH2)5Tyr(Me)AVP) binding to V1 AVP receptors in the three different cell types used. The order of potency was ethylisopropyl amiloride (EIPA) greater than Benzamil greater than amiloride. AVP mobilization of intracellular calcium was blocked by the V1 antagonist d(CH2)5Tyr(Me)AVP and was reduced by EIPA in a dose-dependent manner. Moreover, EIPA also inhibited prostaglandin F2 alpha mobilization of intracellular calcium. Alkalinization of the intracellular pH with ammonium chloride reversed the inhibitory effect of EIPA but not that of the V1 antagonist on AVP-induced calcium mobilization. Both amiloride and EIPA blocked AVP-induced aggregation of human platelets and contraction of mesangial cells and glomeruli preparations independently of receptor site antagonism. In conclusion, amiloride analogues interfere with activation of V1 vascular receptors by AVP at different levels including binding to the receptor site, mobilization of intracellular calcium, cell contraction or aggregation, and presumably alteration of intracellular ion transports.

Mechanisms of vasopressin-induced increase in intracellular Na+ in vascular smooth muscle cells.

The present study was undertaken to examine the interrelationship between the arginine vasopressin (AVP)-induced dynamic changes in intracellular Ca2+ and Na+ concentrations in cultured rat aortic vascular smooth muscle cells (VSMC) by the direct measurements of intracellular Na+ concentration [( Na+]i), cytosolic free calcium [( Ca2+]i), and intracellular pH (pHi) using fluorescence dyes. AVP increased [Ca2+]i and [Na+]i in a dose-dependent manner; the rise in [Ca2+]i preceded the rise in [Na+]i. Pretreatment with the V1 antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylenepropionic acid) 2-(O-methyl)-tyrosine] AVP [( d(CH2)5Tyr(Me)]AVP) completely blocked the effects of AVP on [Ca2+]i and [Na+]i. The V2 agonist 1-desamino-8-D-AVP, DDAVP, did not affect basal [Na+]i or the AVP-induced increase in [Na+]i. Also, Ca(2+)-free solution completely blocked the AVP-induced increase in [Na+]i. Moreover, Ca(2+)-free solution decreased the AVP-induced intracellular acidification and blunted the later AVP-induced intracellular alkalinization. These results therefore indicate that after binding to the V1 receptor AVP increases [Na+]i mediated by the activation of Na(+)-Ca2+ and Na(+)-H+ exchanges in VSMC. All of these cellular events are completely dependent on an increase in cellular Ca2+ uptake produced by AVP.

Autoradiographic localization of oxytocin receptors in the endometrium during the oestrous cycle of the ewe.

The present study was designed to determine the localization of the endometrial oxytocin receptor during the ovine oestrous cycle, particularly on day 14, the time of initiation of luteal regression in the ewe. Samples were obtained from 29 ewes at different stages of the oestrous cycle (several during the luteal phase and on every day between day 14 (-2) and day +3 of the oestrous period). Oxytocin receptors were localized autoradiographically in sections of uterine tissue, using the 125I-labelled oxytocin receptor antagonist [1-(beta-mercapto-beta, beta-cyclopentamethylene propionic acid), 2-(ortho-methyl)-Tyr2,Thr4,Orn8, Tyr9-NH2]-vasotocin (125I-labelled OTA). There was some variation in the pattern of 125I-labelled OTA labeling between different uterine tissue samples from the same ewe and also between samples obtained from different ewes thought to be at the same stage of the oestrous cycle. A clear overall pattern did, however, emerge with 125I-labelled OTA-binding sites distributed between luminal epithelial cells, glandular epithelial cells and caruncular stromal cells to varying extents on different days of the cycle. During the luteal phase (days 5-12) clear specific labelling of endometrial tissue was generally absent. On day 14 labelling was evident on the luminal epithelium, but only in nine tissue samples out of a total of 18 studied, indicating that the entire luminal surface did not contain oxytocin receptors at this time. Between the day before oestrus and day 3 of the oestrous cycle the luminal epithelium was consistently labelled.(ABSTRACT TRUNCATED AT 250 WORDS)

An investigation into the influence of vasopressin on perfusion of the cortex and papilla of the rat kidney

An investigation was undertaken to examine the effects of vasopressin on blood pressure and perfusion of the cortical and papillary regions of the kidney, and to determine the receptor subtype involved. Pentobarbitone-anaesthetized rats were used and laser-Doppler flowmetry applied to measure regional renal haemodynamics. Infusion of vasopressin at 10, 20 and 40 mU kg-1 min-1 caused dose-related increases in blood pressure and reductions in cortical and papillary perfusion of approximately 21, 35 and 41%, respectively at the highest dose. Administration of the V1-receptor antagonist, [1-(beta-mercapto-beta,beta-cyclopentamethylene propionic acid), 2-(o-methyl)tyrosine]-Arg8-vasopressin, at 1 microgram kg-1 plus 5 micrograms kg-1 h-1 or four times this dose had no effect on the basal levels of any variable. Vasopressin administration during the low dose of antagonist increased blood pressure and reduced papillary perfusion, the magnitudes of which were only slightly less than those obtained in the absence of the drug, whereas there was a significant attenuation of the response in cortical perfusion. During infusion of the V1 antagonist at 4 micrograms kg-1 plus 20 micrograms kg-1 h-1, vasopressin had no effect on either blood pressure or renal haemodynamics. Infusion of the V2 antagonist, [d(CH2)5, D-Phe2, Ile4, Arg8, Ala9-NH2]-vasopressin at 1 microgram kg-1 plus 5 micrograms kg-1 h-1, and twice this dose had no effect on the basal value of any variable and had no effect on the ability of vasopressin to induce an increase in blood pressure or cause reductions in renal cortical and papillary perfusions. However, the administration of the V2 antagonist at 4 micrograms kg-1 plus 20 micrograms kg-1 h-1 significantly attenuated blood pressure, cortical and papillary perfusion responses to the vasopressin. These studies have shown that vasopressin, given at doses which increased blood pressure, caused dose-related decreases in perfusion of renal cortex as well as the papilla. The data further show that these systemic and renal actions were mediated primarily by V1-receptors and that the contribution of V2-receptors at these vascular beds was very small.

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(beta-mercapto-beta,beta-cyclopentamethylenepropionic acid), 2-O-methyltyrosine, 4-threonine, 8-ornithine, 9-125I-tyrosylamide]vasotocin [125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT] to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4 degrees C, specific binding sites (expressed at 10(-18) mol 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67 +/- 0.49; cortical thick ascending limbs, 2.20 +/- 0.80; cortical collecting ducts, 2.39 +/- 0.86; outer medullary collecting ducts (OMCD), 2.54 +/- 0.53 and inner medullary collecting ducts, 5.33 +/- 0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific 125I-d(CH2)5[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4 degrees C were 1.06 x 10(7) M-1 min-1 and 1.95 x 10(-2) min-1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:dDAVP greater than AVP greater than d(CH2)5-[Tyr (Me)2, Thr4, Tyr-NH(2)9]OVT = AVT = OT greater than d(CH2)5[Tyr(Me)2]AVP = [Thr4, Gly7]OT greater than [Phe2, Orn8]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V2 vasopressin receptors.