Insulin receptors along the rat nephron: [125I] Insulin binding in microdissected glomeruli and tubules

Binding of [¹²⁵I] Tyr A¹⁴ human insulin ([¹²⁵I] insulin) was measured at 4°C in glomeruli and pieces of tubule microdissected from collagenase-treated rat kidneys. For glomeruli and all segments tested, total and non specific binding increased linearly with glomeruli number or tubular length. When determined with 4.0 nM labelled hormone, the distribution of specific binding sites (expressed as 10^–18 mol [¹²⁵I] insulin bound per glomerulus or mm tubule length) was as follows: glomerulus, 2.5±0.3; proximal convoluted tubule (PCT), 12.6±0.6; pars recta (PR), 4.0±2.3; thin descending limb (TDL), 0.6±0.2; thin ascending limb (TAL), 0.6±0.2; medullary thick ascending limb (MAL), 0.8±0.1; cortical ascending limb (CAL), 2.1±0.1; distal convoluted tubule (DCT), 5.6±1.1; cortical collecting tubule (CCT), 3.2±0.3 and outer medullary collecting tubule (MCT), 2.3±0.1. Specific [¹²⁵I] insulin binding to glomeruli and tubule segments was time- and dose-dependent, saturable, reversible after elimination of free labelled ligand, and inhibited by unlabelled human insulin. When analysed in Scatchard and Hill coordinates, the binding data revealed a negative cooperation in the interaction processes between [¹²⁵I] insulin and glomerular and tubular binding sites, with apparent dissociation constants and Hill coefficients of the following values: glomerulus, 0.6 nM and 0.60; PCT, 10.0 nM and 0.55; MAL, 4.3 nM and 0.80; CAL, 2.0 nM and 0.74; CCT, 7.6 nM and 0.80 and MCT, 1.0 nM and 0.57 respectively. The stereospecificity of nephron binding sites was assessed in competitive experiments showing that unlabelled bovine and procine insulins were as efficient as human insulin for displacing [¹²⁵I] insulin, whereas A and B chains of insulin and unrelated peptide hormones were almost inactive. These results indicate that the detected [¹²⁵I] insulin binding sites may correspond to physiological insulin receptors.Abbreviations used [¹²⁵I] Insulin [¹²⁵I] Tyr A¹⁴ human insulin - PCT proximal convoluted tubule - PR pars recta - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT outer medullary collecting tubule     

Dopamine inhibits Na/K-ATPase in single tubules and cultured cells from distal nephron

Dopamine decreases tubular sodium reabsorption, attributed in part to Na/K-ATPase inhibition in the proximal convoluted tubule (PCT). Because the final regulation of sodium excretion occurs in the collecting duct, where we have demonstrated specific dopamine DA₁ binding sites, we examined the effects of dopamine, and of DA₁ and DA₂ receptor agonists on the Na/K pump in the microdissected rat cortical collecting duct (CCD) and in Madin-Darby canine kidney (MDCK) cells, a line derived from the dog distal nephron. Dopamine inhibited pump activity in CCD by approximately 40%–50%, an effect proportionally larger than in the PCT. Unlike in the latter, the effect of dopamine was reproduced by the DA₁ agonist fenoldopam, which inhibited the CCD pump in dose-dependent manner (maximum, 10 M). The DA₂ agonist quinpirole was without effect, either alone or in combination with fenoldopam. These actions on Na/K-ATPase paralleled in reciprocal fashion effects on adenylate cyclase: dopamine or fenoldopam, but not quinpirole, produced a significant increase in cAMP content, and the stimulation by dopamine was blocked by SCH 23390. Inhibitors of cAMP phosphodiesterase (3-isobutyl-1-methylxanthine and theophylline), as well as forskolin and dibutyryl-cAMP, mimicked the effect of dopamine on the pump, underscoring the role of increased cAMP in this phenomenon. Both dopamine and fenoldopam inhibited Na/K-ATPase activity in MDCK cells. The results indicate that besides the PCT dopamine inhibits Na/K-ATPase activity in cells of the distal nephron, where its effect on the pump appears to be more pronounced and is mediated by activation of the DA₁ receptor. The natriuretic effect of dopamine is probably exerted at both proximal and distal nephron sites.     

Negative crosstalk between M1 and M2 muscarinic autoreceptors involves endogenous adenosine activating A1 receptors at the rat motor endplate

At the rat motor nerve terminals, activation of muscarinic M₁ receptors negatively modulates the activity of inhibitory muscarinic M₂ receptors. The present work was designed to investigate if the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involved endogenous adenosine tonically activating A₁ receptors on phrenic motor nerve terminals. The experiments were performed on rat phrenic nerve-hemidiaphragm preparations loaded with [³H]-choline (2.5 μCi/ml). Selective activation of muscarinic M₁ and adenosine A₁ receptors with 4-(N-[3-clorophenyl]-carbamoyloxy)-2-butyryltrimethylammonium (McN-A-343, 3 μM) and R-N⁶-phenylisopropyladenosine (R-PIA, 100 nM), respectively, significantly attenuated inhibition of evoked [³H]-ACh release induced by muscarinic M₂ receptor activation with oxotremorine (10 μM). Attenuation of the inhibitory effect of oxotremorine (10 μM) by R-PIA (100 nM) was detected even in the presence of pirenzepine (1 nM) blocking M₁ autoreceptors, suggesting that suppression of M₂-inhibiton by A₁ receptor activation is independent on muscarinic M₁ receptor activity. Conversely, the negative crosstalk between M₁ and M₂ autoreceptors seems to involve endogenous adenosine tonically activating A₁ receptors. This was suggested, since attenuation of the inhibitory effect of oxotremorine (10 μM) by McN-A-343 (3 μM) was suppressed by the A₁ receptor antagonist, 1,3-dipropyl-8-cyclopentylxanthine (2.5 nM), and by reducing extracellular adenosine with adenosine deaminase (0.5 U/mL) or with the adenosine transport blocker, S-(p-nitrobenzyl)-6-thioinosine (NBTI, 10 μM). The results suggest that the negative crosstalk between muscarinic M₁ and M₂ autoreceptors involves endogenous adenosine outflow via NBTI-sensitive (es) nucleoside transport system channelling to the activation of presynaptic inhibitory A₁ receptors at the rat motor endplate.     

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-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 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° 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° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–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>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[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.     

Glucagon receptors along the nephron: [125I]glucagon binding in rat tubules

Binding of [¹²⁵I]glucagon was measured in microdissected pieces of tubules from the rat nephron. Specific glucagon binding sites were found only in nephron segments containing a glucagon-sensititive adenylate cyclase activity. At 7.5 nM labelled hormone, higher levels of specific binding (16–27×10^–18 mol mm^–1) were found in the thick ascending limb of the Henle's loop and in the distal convoluted tubule and lower binding levels (2–5×10^–18 mol mm^–) in the collecting tubule whereas specific binding could not be detected in the proximal tubule and in the thin segments of the Henle's loop. In the medullary thick ascending limb, Scatchard analysis of specific [¹²⁵I]glucagon binding indicated an apparent equilibrium dissociation constant of 2.4 nM. The stereospecificity of binding sites in medullary thick ascending limbs and medullary collecting tubules, was assessed by competition experiments using unlabelled glucagon, enteroglucagon and unrelated hormones (vasopressin, calcitonin, parathyroid hormone and insulin); in both segments, glucagon was more active than enteroglucagon in displacing labelled glucagon from its tubular binding sites, whereas all other hormones tested were inactive. These results indicate that tubule binding sites might be the physiological receptors for glucagon involved in adenylate cyclase activation.Abbreviations PCT proximal convoluted tubule - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL cortical ascending limb - DCT distal convoluted tubule - CCT cortical collecting tubule - MCT medullary collecting tubule     

Effects of prolactin on Na−K-ATPase activity along the rat nephron

To test prolactin (PRL) action on osmoregulation in mammals, we evaluated in the rat the effect of this hormone on a major enzyme in renal regulation of water and electrolyte: renal Na–K-ATPase. Enzyme activity was determined by cytochemistry in medullary ascending limb (MAL) and distal convoluted tubule (DCT) from rats treated either by bromocriptine, or by PRL. Three hours after a bromocriptine injection (0.1 mg/100 g IP) a significant decrease of Na–K-ATPase activity is observed in both MAL (80% of control values,p<0.001) and DCT (78%,p<0.01). Reciprocally, a significant (p<0.001) increase in enzyme activity is induced 3 h after a single PRL injection (140 g/100 g IM), in both segments (MAL: 165%, DCT: 172% of control activities) and persists 6 h after the injection (MAL: 130%, DCT: 118%). Na–K-ATPase activity was correlated to plasma PRL levels (r=0.78 in DCT,r=0.89 in MAL). A direct effect of PRL on the tubule is suggested by results from experiments in which PRL, at various concentrations, is added in vitro on renal slices before Na–K-ATPase activity measurements. The increase in Na–K-ATPase activity exhibits a log-dose dependency with PRL concentration (p<0.01) and is still observed when AVP antagonist is added before PRL incubation, ruling out the possible role of AVP contamination of PRL. These results suggest a direct effect of PRL on renal Na–K-ATPase in MAL and DCT.     

Atrial natriuretic peptide receptors along the rat and rabbit nephrons: [125I] α-rat atrial natriuretic peptide binding in microdissected glomeruli and tubules

Binding of [¹²⁵I] -rat atrial natriuretic peptide ([¹²⁵I] -RANP) was measured in glomeruli and pieces of tubule microdissected from rat and rabbit nephrons. High densities of specific ANP binding sites were found only in the glomeruli (10–30×10^–18 mol·glom^–1), whereas no specific binding could be detected in the proximal tubule, the thin segments of the Henle's loop, the thick ascending limb, the distal tubule and the cortical and outer medullary collecting tubules. Rising the temperature from 4° C to 35° C resulted in biphasic kinetics of binding, suggesting a temperature-dependent inactivation of labelled hormone by glomeruli. At 4° C, specific binding of [¹²⁵I] -RANP was time and dose-dependent and Scatchard analysis of data indicated an apparent equilibrium dissociation constant of 0.63 nM. Competition experiments revealed the following sequence of stereospecificity for binding to rat glomeruli: RANP 3–28>[¹²⁵I] -RANP=[¹²⁵I] -HANP=-RANP=atriopeptin III > atriopeptin II, whereas binding was unaffected by pharmacological doses of unrelated peptide hormones, prostaglandins, adrenergic agonists, dopamine, histamine and carbamylcholine. The results indicate that glomerular binding sites might be the physiological ANP receptors.Abbreviations ANP atria natriuretic peptide - 0RANP alpha rat atrial natriuretic peptide - PCT proximal convoluted tubule - PR pars recta - TDL thin descending limb - TAL thin ascending limb - MAL medullary thick ascending limb - CAL dortical thick ascending limb - DCT distal convoluted tubule - DCTb distal convoluted tubule bright - DCTg distal convoluted tubule granular - CCT conrtical collecting tubule - MCT outer medullary collecting tubule     

Effects of calcium-sensing receptors on apoptosis in rat hippocampus during hypoxia/reoxygenation through the ERK1/2 pathway

Objectives: To explore the effects of calcium-sensing receptors (CaSR) on apoptosis in rat hippocampus during hypoxia/reoxygenation (H/R). Methods: After rat hippocampus was isolated, the cultures were subjected to H/R, and meanwhile gadolinium chloride (GdCl₃, agonist of CaSR) and NPS 2390 (antagonists of CaSR) were added to reperfusion solution. The number of hippocampal neuron, cell viability and apoptosis rate were determined by inverted microscope, 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and flow cytometer (FCM), respectively. Besides, caspase-3, Bax, cytochrome C (Cyt-c), extracellular signal-regulated protein kinase (ERK) 1/2, pERK1/2, P38 and pP38 were analyzed by Western blotting. Results: The hippocampal neuron number and cell viability were significantly decreased during H/R, and were further significantly reduced when co-treatment with CaSR agonist GdCl₃. But the effects of GdCl₃ were attenuated by NPS-2390. Whereas, apoptosis rate, the expression level of caspase-3, Bax and Cyt-c were all significantly increased under H/R condition, and was further significantly increased by GdCl₃, but were reversed by NPS-2390 (P < 0.05). Moreover, there were no significant differences in expression of ERK1/2, P38 and pP38 among different groups. However, the expression of pERK1/2 was significantly increased during H/R, but was significantly reduced by NPS 2390 (P < 0.05). Conclusion: The results suggest that CaSR might play significant roles in the induction of hippocampus apoptosis in rat during H/R through phosphorylation of ERK1/2.     

Interaction between dopamine D1 and D2 receptors in modulation of the immune response

The interaction between dopamine D₁ and D₂ receptors plays a role in immunomodulation. The results of thus interaction depends on the degree of receptor activation with selective agonists in different doses. Combined treatment with agonists of D₁ and D₂ receptors in high doses had a synergistic effect in the mechanisms of immunomodulation. Receptor agonists in low doses suppressed the immune response. Our results suggest that weak activation of one of these receptors is accompanied by inactivation of the other receptor type. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 141, No. 5, pp. 488–490, May, 2006     

Hypoxia-induced desensitization and internalization of adenosine A1 receptors in the rat hippocampus

Activation of A1 adenosine receptors is important for both the neuromodulatory and neuroprotective effects of adenosine. However, short periods of global ischemia decrease A1 adenosine receptor density in the brain and it is not known if a parallel loss of functional efficiency of A1 adenosine receptors occurs. We now tested if hypoxia leads to changes in the density and efficiency of A1 adenosine receptors to inhibit excitatory synaptic transmission in rat hippocampal slices. In control conditions, the adenosine analog 2-chloroadenosine, inhibited field excitatory post-synaptic potentials with an EC50 of 0.23 microM. After hypoxia (95% N2 and 5% CO2, for 60 min) and reoxygenation (30 min), the EC50 increased to 0.73 microM. This EC50 shift was prevented by the presence of the A1 adenosine receptor antagonist 8-phenyltheophyline, but not by the A(2A)R antagonist 7-(2-phenylethyl)-5-amino-2-(2-furyl)-pyrazolo-[4,3-e]-1,2,4-triazolo[1,5-c] pyrimidine, during the hypoxic period. This decreased efficiency of A1 adenosine receptors was not paralleled by a global change of A1 adenosine receptor density or affinity (as evaluated by the binding parameters obtained in nerve terminal membranes). However, the density of biotinylated A1 adenosine receptors at the plasma membrane of nerve terminals was reduced by 30% upon hypoxia/reoxygenation, in a manner prevented by the A1 adenosine receptor antagonist 1,3-dipropyl-8-cyclopentylxanthine and mimicked by prolonged (60 min) supra-maximal activation of A1 adenosine receptors with 2-chloroadenosine (10 microM). These results indicate that hypoxia leads to a rapid (<90 min) homologous desensitization of A1 adenosine receptor-mediated inhibition of synaptic transmission that is likely due to an internalization of A1 adenosine receptors in nerve terminals.     

Presynaptic D1 dopamine receptors facilitate glutamatergic neurotransmission in the rat globus pallidus

The effects of D1/5 dopamine agonists on spontaneous excitatory postsynaptic currents (sEPSCs) were studied in neurons of the rat globus pallidus using whole-cell recordings in the presence of TTX and bicuculline. In this condition, CNQX abolished the sEPSCs, indicating that they were solely mediated by AMPA receptors. SKF 38393, a D1-like agonist, increased the frequency but not the amplitude of the sEPSCs, suggesting a presynaptic site of action. The increase in frequency was blocked by SCH 23390, a D1/5 antagonist. Quinpirole, a D2-like agonist, decreased the frequency but did not affect the amplitude of the synaptic currents. SKF 38393 increased the frequency of sEPSCs currents, even in presence of quinpirole, indicating that D1/5- and D2-like receptors independently modulate glutamate release upon a single neuron. The results suggest that the dopaminergic control of the glutamate transmission in the globus pallidus may play a role in processing cortical information in the indirect pathway of the basal ganglia.     

Contribution of 5-HT1A serotonin receptors of the brain to the regulation of hereditary catalepsy

Selective agonists 5-HT_1A of serotonin receptors (8-OH-DPAT and flezinoxan) had an inhibitory effect on the manifestation of hereditary catalepsy in mice and rats. No differences were revealed in specific binding of³H-8-OH-DPAT to 5-HT_1A receptors in the striatum of either cataleptic or noncataleptic mice and rats. Nonetheless, an increase of the density of these receptors was observed in the frontal cortex of CBA mice predisposed to catalepsy in comparison with mice of the noncataleptic C57Bl strain. The data indicate a contribution of 5-HT_1A receptors to the regulation of hereditary catalepsy. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 118, N ^o 12, pp. 633–635, December, 1994 Presented by V. P. Kaznacheev, Member of the Russian Academy of Medical Sciences     

Caffeine reverses antinociception by oxcarbazepine by inhibition of adenosine A1 receptors: Insights using knockout mice

Oxcarbazepine is an anticonvulsant drug that has been explored as a novel therapeutic agent to treat neuropathic pain in humans. It produces antinociception in several preclinical models of pain, and these actions are blocked by methylxanthine adenosine receptor antagonists which implicates adenosine it its actions. In this study, the antinociceptive effect of oxcarbazepine, and the ability of caffeine to reverse its actions, were examined using the formalin test (2%) in wild-type mice and in mice lacking adenosine A₁ receptors by way of further exploring the involvement of adenosine in its actions. Oxcarbazepine produced dose-related suppression of formalin-evoked flinching responses in wild-type mice following both systemic and intraplantar administration, and this action was reversed by systemic and intraplantar administration of caffeine, respectively. The ability of oxcarbazepine to inhibit flinching after systemic and intraplantar administration was unaltered in homozygous (−/−) and heterozygous (+/−) adenosine A₁ receptor knockout mice. However, caffeine no longer reversed this antinociception. Our results indicate that, while adenosine A₁ receptors are not required for oxcarbazepine to produce antinociception in knockout mice, such receptors are essential in order to see caffeine reversal of this antinociceptive effect.     

Involvement of dopamine D<Subscript>1</Subscript> and D<Subscript>2</Subscript> receptors in the rat nucleus accumbens in immunostimulation

Analysis of the nature of changes in the immune response in operated Wistar rats showed that electrolytic lesioning of the nucleus accumbens, the site of the greatest density of dopamine D₁ and D₂ receptors, led to suppression of the immune response in animals immunized with sheep erythrocytes. Administration of SKF 38393 (20 mg/kg) and quinpirol (1 mg/kg), selective agonists of dopamine D₁ and D₂ receptors respectively, to sham-operated rats induced significant increases in immune responses. However, no immunostimulation was seen on administration of the selective dopamine D₂ agonist quinpirol to animals with lesions to the nucleus accumbens as compared with controls. At the same time, treatment of animals with nucleus accumbens lesions using the dopamine D₁ receptor agonist SKF 38393 had no effect on the immune response as compared with that in sham-operated animals given the D₁ receptor agonist. These data provide evidence that dopamine D₂ receptors in the nucleus accumbens have a role in the mechanisms of immunostimulation, though D₂ receptors in other brain structures may also make some contribution to this process; D₁ receptors in the nucleus accumbens make no significant contribution to controlling the immune response. __________ Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 91, No. 11, pp. 1281–1287, November, 2005.     

[Arginine]vasopressin hydrolyses phosphoinositides in the medullary thick ascending limb of mouse nephron

NaCl reabsorption across the thick ascending limb of Henle's loop (TAL) is stimulated by several hormones, in particular vasopressin acting through V₂ receptors and cyclic AMP production. This study used suspensions of medullary TAL (mTAL) tubules from the mouse nephron to investigate the possibility that, besides activating adenylyl cyclase, vasopressin also stimulates phospholipase C via V₁ receptor occupancy. Two different methods, phosphoinositide labelling and inositol trisphosphate (InsP ₃) radioimmunoassay, were used to show that [arginine]vasopressin (AVP) rapidly stimulated the formation of InsP ₃, which peaked at 200%–250% of control within the first minute of incubation with 10 nmol/l vasopressin at 37°C, and declined to basal level after 5–10 min. Dose/response curves for InsP ₃, established at 30°C and 37° C using radioimmunoassay, showed a half-maximal stimulation of InsP ₃ production at about 1 nmol/l AVP and a maximal response at 10 nmol/l. Similar values were obtained for the response to AVP in terms of cAMP accumulation. InsP ₃ content in the presence of higher concentrations of AVP (1 mol/l) was significantly lower (P<0.001) than in the presence of 10 nmol/l AVP, giving a bell-shaped appearance to the dose/response curve at 37° C but not at 30° C. The V₂ receptor agonist, 1-deamino-[8DArg]vasopressin (dAVP) did not stimulate the formation of InsP ₃, and the V₁ receptor antagonist d(CH₂)₅[Tyr(Me)²]AVP inhibited AVP-induced InsP ₃ formation, which therefore appeared to be mediated by V₁ receptor occupancy. Under the same conditions, AVP also induced the formation of diradylglycerol via V₁ receptor activation, with an analogous dose/response curve. These results show that AVP, in addition to its well-known action through V₂ receptors, also acts on the mouse mTAL through a V₁-mediated stimulation of phospholipase C. Cyclic AMP controls this transduction pathway: dAVP (10 nmol/l), dibutyryl-cAMP (1 mmol/l and 0.1 mmol/l) and forskolin (1 mol/l) decreased the InsP ₃ formation induced by AVP. Dibutyryl-cAMP itself at 37°C also reduced the diglyceride content.     

Caffeine reverses antinociception by amitriptyline in wild type mice but not in those lacking adenosine A1 receptors

Amitriptyline is used to treat neuropathic pain in humans. It produces antinociception in several animal models of pain, and this effect is blocked by methylxanthine adenosine receptor antagonists which implicates adenosine it its actions. Here, the antinociceptive effect of amitriptyline, and the ability of caffeine to reverse it, were examined using the formalin test (a model of persistent pain) in wild type mice and mice lacking the adenosine A₁ receptor (A1R). Amitriptyline produced dose-related suppression of flinching in wild type mice following both systemic and intraplantar drug administration; both of these effects were unaltered in A1R −/− mice. Following systemic administration, caffeine reversed the systemic effect of amitriptyline in wild type, but not A1R −/− mice; −/+ mice exhibited an intermediate effect. Intraplantar administration of caffeine also reversed the effect of intraplantar amitriptyline in A1R +/+, but not in −/− or +/− mice. These results indicate that adenosine A₁ receptors are not required in order for amitriptyline to cause antinociception in mice, but they are required to see caffeine reversal of this antinociceptive effect. When A1Rs are present, actions of amitriptyline may, however, partly depend on A1Rs.     

Instantaneous depolarization of T cells via dopamine receptors,and inhibition of activated T cells of Psoriasis patients and inflamed human skin,by D1-like receptor agonist: Fenoldopam

Activated T cells are pathological in various autoimmune and inflammatory diseases including Psoriasis, and also in graft rejection and graft-versus-host-disease. In these pathological conditions, selective silencing of activated T cells through physiological receptors they express remains a clinical challenge. In our previous studies we found that activation of dopamine receptors (DRs) in resting human T cells activates these cells, and induces by itself many beneficial T cell functions. In this study, we found that normal human T cells express all types of DRs, and that expression of D1R, D4R and D5R increases profoundly after T cell receptor (TCR) activation. Interestingly, DR agonists shift the membrane potential (V_m) of both resting and activated human T cells, and induces instantaneous T cell depolarization within 15 seconds only. Thus, activation of DRs in T cells depolarize these immune cells, alike activation of DRs in neural cells. The skin of Psoriasis patients contains 20-fold more D1R⁺ T cells than healthy human skin. In line with that, 25-fold more D1R⁺ T cells are present in Psoriasis humanized mouse model. Highly selective D1-like receptor agonists, primarily Fenoldopam (Corlopam) – a D1-like receptor agonist and a drug used in hypertension, induced the following suppressive effects on activated T cells of Psoriasis patients: reduced chemotactic migration towards the chemokine SDF-1/CXCL12; reduced dramatically the secretion of eight cytokines: tumor necrosis factor-α, interferon-γ, interleukin-1β (IL-1β), IL-2, IL-4, IL-6, IL-8 and IL-10; and reduced three T cell activation proteins/markers: CD69, CD28 and IL-2. Next, we invented a novel topical/dermal Fenoldopam formulation, allowing it to be spread on, and providing prolonged and regulated release in, diseased skin. Our novel topical/dermal Fenoldopam: reduced secretion of the eight cytokines by activated human T cells; reduced IL-1β and IL-6 secretion by human lipopolysaccharide-inflamed skin; eliminated preferentially >90% of live and large/proliferating human T cells. Together, our findings show for the first time that both resting and activated T cells are depolarized instantaneously via DRs, and that targeting D1-like receptors in activated T cells and inflamed human skin by Fenoldopam, in Psoriasis, and potentially in other T cell-mediated diseases, could be therapeutic. Validation in vivo is required.     

Re-evaluation of the expression of the gastric H,K-ATPase α subunit along the rat nephron

 Under normal conditions, the rat collecting duct displays an H,K-ATPase activity with kinetic and pharmacological properties very close to those of the gastric H,K-ATPase. However, whether the collecting duct H,K-ATPase and the gastric enzyme are identical remains controversial. Therefore, we re-evaluated the expression of the mRNAs encoding the gastric H,K-ATPase α subunit in the rat nephron. For this purpose, gastric H,K-ATPase mRNAs were quantitated by RT-PCR at the level of microdissected nephron segments using known amounts of gastric H,K-ATPase cRNA as external standards. Results indicate that gastric H,K-ATPase mRNAs are undetectable (<1 copy per cell) in the glomerulus and along the proximal tubule, thick ascending limb of Henle’s loop and collecting duct, although a faint expression (≈ 400 copies per μg total RNA) is measurable in whole–kidney preparations. Gastric H,K-ATPase mRNA is also absent along the nephron of K-depleted rats and of rats with chronic metabolic acidosis and alkalosis. Taken with other data from the literature, these results suggest that the collecting duct of normal rats might express an H,K-ATPase similar, but not identical, to the gastric isoform. Received: 29 October 1996 / Accepted: 21 November 1996     

Somatostatin modulates dopamine release in rat retina

The aim of the present study was to determine the possible role of somatostatin as a modulator of dopamine release in rat retina. Basal release of dopamine, and how this is influenced by somatostatin receptor (sst) selective ligands, was examined ex vivo in rat retinal explants. Dopamine levels were quantified by high-pressure liquid chromatography (HPLC) with electrochemical detection. Basal levels of dopamine were measured over 120 min of tissue incubation and found to be 1.17+/-0.35 ng/ml. Somatostatin (10(-6), 10(-5), 10(-4)M) increased dopamine levels in a concentration-dependent manner, while the sst(2) antagonist CYN154806 (10(-4)M) reversed its actions. BIM23014 (sst(2) agonist) increased dopamine levels in a statistically significant manner only at the concentration of 10(-5)M. The sst(1) agonist L797.591 (10(-5), 10(-4)M) also increased dopamine levels, while activation of the sst(3) receptor (sst(3) agonist, L796.778, 10(-4)M) had no effect. These data substantiate a neuromodulatory role for sst(1) and sst(2) somatostatin receptors in the retina and show for the first time somatostatin's influence on dopamine release.