Biomedical subjects

R A Felder

Publications and source records attributed to R A Felder.

At least 55 records · Page 3Linked to original sources

PubMed1996

High-level expression of rat D1A dopamine receptor cDNA in mouse fibroblast LTK- cells by n-butyrate.

1. In order to develop a simple, efficient system for the high-level expression of dopamine receptors in eukaryotic cells, we have studied the effects of n-butyrate on the expression of rat D1A dopamine receptor cDNA in mouse fibroblast LTK- cells as compared with those of n-butyrate on endogenous D1 receptor levels in opossum kidney cells. 2. In the transfected LTK- cell membranes with pRc/CMV-D1A receptor cDNA, a selective D1 dopamine antagonist, [3H]-SCH 23390, exhibited a Kd of 0.9 +/- 0.1 nmol/L and a Bmax of 0.35 +/- 0.05 pmol/mg protein (n = 5). 3. Addition of n-butyrate (2-10 mmol/L) to the culture medium for 48 h dose-dependently increased the D1A receptor level up to 1.5 +/- 0.3 pmol/mg protein (n = 7), although the Kd values were not affected. The increase in receptor level was accompanied by an elevation of selective D1 agonist-induced adenylyl cyclase activity. 4. In contrast, n-butyrate treatment (2-10 mmol/L) did not affect either endogenous D1 receptor levels or fendoldopam-induced adenylyl cyclase activity in opossum kidney cells. 5. These results suggest n-butyrate is a useful tool for obtaining high-level expression of D1A dopamine receptor cDNA in mouse fibroblast LTK- cells.

Adenylyl Cyclases↗

PubMed1996

Expression of the subtype 1A dopamine receptor in the rat heart.

The subtype 1A dopamine receptor (D1A) has recently been detected in the rat kidney. In the present study using light microscopic immunohistochemistry, electron microscopic immunocytochemistry, and in situ amplification of mRNA, we demonstrate the D1A receptor in Sprague-Dawley and Wistar Kyoto rat hearts. For immunohistochemistry and immunocytochemistry, anti-peptide polyclonal antibodies were directed toward amino acid sequences of the third extracellular and intracellular domains of the native receptor. Selectivity was validated by recognition of the D1A receptor expressed in stably transfected LTK- cells. D1A receptor mRNA was detected with a novel transcription-based isothermal in situ amplification system as well as with reverse transcription-polymerase chain reaction. D1A receptor protein was distributed throughout the atrium and ventricular myocardium. Preimmune and preabsorption controls were negative. Electron microscopic immunocytochemistry using the protein A gold method demonstrated the D1A receptor along the cellular membranes of coronary smooth muscle cells and ventricular myocytes and in the myosin thick filaments and M-lines. D1A receptor mRNA was present in coronary vessels and myocardium in amplified but not in unamplified sections. Western blot analysis showed specific D1A bands in transfected LTK- cells and the atrium but not in nontransfected LTK- cells and the ventricle. The selective D1-like receptor agonist SKF38393 stimulated adenylyl cyclase in ventricular myocardial plasma membranes in a dose-related fashion, and the response was abolished by the selective D1-like receptor antagonist SCH23390. These results demonstrate that the D1A receptor gene and protein are expressed in normal rat heart. The physiological and pathophysiological roles and predominant cell signaling mechanism or mechanisms of this receptor remain to be determined.

Animals↗

PubMed1996

Clinical trials of a novel centrifugation method: axial separation.

Specimen centrifugation is one of the most time-consuming tasks associated with preanalytical specimen processing. We report the first clinical use and impact study of the novel centrifugation method, axial separation, using the Axial Separation Module (ASM). The ASM centrifuges medical specimens one at a time (in 1 min 9 s) in proprietary Axial Process Containers (APC). Here we report tests of the ASM in an outpatient clinical laboratory. Use of the ASM in the outpatient clinic laboratory led to an average total improvement (decrease in total bench time) of 8 min 9 s per specimen compared with conventional centrifugation. Laboratory analysis revealed no statistical difference between plasma obtained from the APC vs that from a Becton Dickinson Vacutainer Tube. We conclude the ASM offers a cost-effective alternative to conventional centrifugation. Further, the use of axial separation may allow specimen separation to become an integral part of the phlebotomy process.

Blood↗

PubMed1996

Dopamine D2L receptors stimulate Na+/K(+)-ATPase activity in murine LTK- cells.

Ion transport can be regulated by dopamine receptors. D1-like receptors inhibit both Na+/H+ exchange (NHE) and Na+/K(+)-ATPase activity, whereas D2-like receptors stimulate NHE. However, the effect of D2-like receptors on Na+/K(+)-ATPase activity is controversial. In renal proximal tubular cells, where several D1-like and D2-like receptors are expressed, D2 agonists have been reported either to have no effect or to act in concert with D1 agonists to inhibit Na+/K(+)-ATPase activity. We therefore studied the effect of D2 receptors on Na+/K(+)-ATPase activity in LTK- cells transfected with a rat D2Long receptor cDNA (maximum receptor density = 0.91 +/- 0.26 pmol/mg protein, dissociation constant = 2.39 +/- 0.79 nM, seven experiments). The activation of D2 receptors in these transfected cells by the selective D2 agonist LY171555 led to the inhibition of forskolin-stimulated cAMP accumulation. In the D2Long-transfected, but not in nontransfected cells, LY171555 caused a concentration-dependent stimulation of Na+/K(+)-ATPase activity (EC50 = 0.55 +/- 0.2 microM, Emax = 28 +/- 6%, six experiments), which was completely blocked by the D2-selective antagonist (-)-sulpiride. The D2-stimulated Na+/K(+)-ATPase activity was not secondary to D2 receptor activation of K+ channels or NHE activity since LY171555 stimulated Na+/K(+)-ATPase activity in D2Long-transfected cells, even when K+ channels were blocked by CsCl and intracellular Na+ was clamped by monensin. The D2-stimulated Na+/K(+)-ATPase activity was blocked by pertussis toxin and mimicked by dideoxyadenosine. We conclude that agonist occupancy of D2Long dopamine receptors stimulates Na+/K(+)-ATPase activity; this effect is mediated by the inhibition of cAMP production and is independent of intracellular Na+ and K+ concentration.

Adenylyl Cyclases↗

PubMed1996

Robotics and the changing face of the clinical laboratory.

Rapid changes in healthcare coupled with parallel advances in technology have stimulated the evolution of new approaches for laboratory automation. In particular, the emergence of commercially available laboratory robotic systems offers promise for streamlining the clinical laboratory. Increasing cost-containment pressures make the application of this technology extremely attractive, and several organizations have begun to systematically integrate robotic devices into their laboratory automation schemes. Integration of these technologies, however, presents many challenges for software developers, instrument manufacturers, and laboratory workers. Differing needs across laboratories require flexibility and intelligence in robots, instruments, and control systems. Standardization of mechanical and electronic interfaces will be key to making these systems easy to integrate. Systems engineering, aided by simulation modeling and artificial intelligence schemes, will be important to assist in the design of optimal configurations. Software for the overall control of integrated automation will be needed that can be tailored by the laboratorian to fit the requirements of the individual laboratory. Thus, laboratory workers will need to be actively involved in implementing this new wave of laboratory automation, becoming well-versed in computers, electronics, and systems engineering.

Autoanalysis↗

PubMed1995

Alpha 1B-adrenergic receptors in rat renal microvessels.

Although several alpha-adrenergic receptor genes are expressed in the rat kidney, their expression in the renal vasculature has not been studied. Since pharmacological studies have suggested that an alpha 1B-adrenergic receptor may mediate renal vasoconstriction, we studied the expression of alpha 1B-adrenergic receptors in renal microvessels, from 10- to 14-week-old male spontaneously hypertensive rats (SHR) and their normotensive control, the Wistar-Kyoto rat (WKY). In these microvessels, isolated by perfusion with iron, alpha 1B-adrenergic receptor mRNA levels (by ribonuclease protection assay) were similar in SHR and WKY rats. Photo-affinity labeling with [125I]-arylazidoprazosin demonstrated the presence of alpha 1B-adrenergic receptor protein. Maximum receptor density (determined by 3H-prazosin binding: Bmax 59.8 +/- 4.1 and 58.7 +/- 4.3; Kd 0.48 +/- 0.05 nM and 0.31 +/- 0.06 nM in SHR and WKY, respectively) and chloroethylclonidine (CEC)-sensitive binding sites (determined by [125I]-(2-beta(4-hydroxyphenyl)-ethylaminomethyl)-tetralone binding) (125I-HEAT) were similar in SHR and WKY rats. There are two novel findings in these studies: (1) the alpha 1B-adrenergic receptor gene is expressed in renal microvessels of WKY and SHR; (2) alpha 1B-adrenergic receptor gene expression in renal microvessels is not altered in adult SHR. The failure to down-regulate expression of the alpha 1B-adrenergic receptor at the mRNA and protein level in the SHR could result in persistence of alpha 1B-adrenergic receptor effects and contribute to the increased vascular resistance in hypertension.

Affinity Labels↗

PubMed1995

Dopamine-1 receptors in the proximal convoluted tubule of Dahl rats: defective coupling to adenylate cyclase.

We have previously reported a defect in the coupling of the renal dopamine-1 receptor (D1) to adenylate cyclase (AC) in the proximal convoluted tubule (PCT) of the spontaneously hypertensive rat (Okamoto-Aoki strain). To determine if this defect is present in another model of hypertension, we microdissected PCTs from Dahl salt-sensitive (DSS) and Dahl salt-resistant (DSR) rats on low- or high-NaCl diet. The ability of two selective D1 agonists, fenoldopam and SND-919-C12, and forskolin to stimulate AC activity in PCT was determined in each of the four groups of rats. Fenoldopam (10(-7) M) and SND-919-C12 (10(-6) M) failed to stimulate AC activity in the PCT of DSS rats whether on a low- or high-NaCl diet. In DSR rats, however, both fenoldopam and SND-919-C12 stimulated AC activity by 289-320% and 220-270%, respectively, whether on a low- or high-NaCl intake. Forskolin (10(-5) M), which directly stimulates AC activity, increased AC activity in all four groups. These studies show that in DSS rats the D1 receptor in the PCT fails to respond to D1 agonists. This defect is not a consequence of the hypertension because it was present in the DSS rats on a low-salt diet and before blood pressure elevation.

Adenylyl Cyclases↗

PubMed1995

Chronic sodium loading augments natriuretic response to acute volume expansion in the preweaned rat.

Positive sodium balance is necessary for normal somatic growth of the neonate, and the neonatal renal response to volume expansion (VE) is attenuated compared with the adult. To test the hypothesis that dietary sodium modulates the developmental response to VE, preweaned rats were artificially reared with either a normal (25 meq/l)- or high-sodium (145 meq/l) diet for 7-8 days and were compared with adult rats receiving normal or high sodium. Serum sodium concentration remained normal in adults on high sodium, whereas neonates became hypernatremic. Glomerular filtration rate (GFR), urinary flow (V), and urinary sodium (UNaV) were measured before and after acute saline VE (1% body wt). While remaining constant in preweaned rats, GFR increased > 50% in adult rats after VE (P < 0.05). High sodium intake augmented V and UNaV after VE but was not sustained in neonates as in adults. Plasma atrial natriuretic peptide (ANP) and guanosine 3',5'-cyclic monophosphate excretion (UcGMPV) were measured, and baseline UcGMPV was lower in preweaned rats receiving normal sodium but increased to levels similar to adult levels after VE. Postexpansion plasma ANP was higher in preweaned rats than in adult rats and was not affected by dietary sodium regardless of age. We conclude that the attenuated postexpansion natriuresis in the neonate is due in part to an adaptive response to limited sodium intake. However, neonatal compensation to increased sodium intake is incomplete and independent of plasma ANP.

Aging↗

PubMed1995

Localization of dopamine D1A receptor protein in rat kidneys.

The dopamine D1A receptor subtype was identified in rat kidney with both light microscopic immunohistochemistry and electron microscopic immunocytochemistry. Antipeptide polyclonal antisera were directed to both extracellular and intracellular regions of the native receptor. The use of such receptor-subtype-selective antibodies allows for the identification of specific dopamine receptor subtype clones that are not distinguished by current pharmacological or receptor-ligand binding technology. Selectivity of the antipeptide antisera was validated by their ability to recognize native receptor protein expressed in permanently transfected mouse LTK- cells. In the rat kidney, D1A receptor protein was localized to the juxtaglomerular apparatus (JGA), proximal tubule, distal tubule, cortical collecting duct, and renal vasculature. In the JGA, the receptor was predominantly located in the arteriolar smooth muscle layer within cytoplasmic granules previously shown to contain renin. In the proximal tubules, staining was localized both on the brush-border and basolateral membranes. The D1A receptor, which is present in the central nervous system, is now identified in the rat kidney at those sites previously labeled as DA1 receptor sites on the basis of pharmacological binding studies. These results suggest that at least some of the renal dopamine DA1 receptors correspond structurally to the central dopamine D1A receptor.

Amino Acid Sequence↗

PubMed1995

Studying the dopaminergic system with transfected receptors.

Dopamine receptors are responsible for diverse effects within and outside the central nervous system. The five dopamine receptors that have been cloned (D1A, D1B also known as D5, D2, D3, and D4) belong to two major families; the D1 like and D2 like dopamine receptors. The D1 like receptors are linked to the stimulation while D1 like receptors are linked to the inhibition of adenylyl cyclase. The physiologic role of the dopamine receptors has been difficult to decipher because several of the subtypes co-exist in the same tissue. However, studies of receptors artificially expressed using transfected cDNA have revealed some of the biochemical mechanisms unique to each dopamine receptor subtype.

Animals↗

PubMed1995

Dopamine D1 receptor regulation of phospholipase C.

Dopamine is an endogenous catecholamine which exerts its actions by occupancy of specific receptors. Dopamine receptors are classified into two main groups: the two cloned D1-like receptors (D1A and D1B in rats; D1B is also known as D5 in humans) are linked to stimulation of adenylyl cyclase, while the three cloned D2-like receptors (D2 or D2A, D3 or D2B, D4 or D2C) are linked to inhibition of adenylyl cyclase. All these dopamine receptors originally cloned from the brain are expressed in tissues outside the central nervous system including the kidney. Dopamine regulates many cellular activities, including transmembrane ion transport. Activation of D1-like receptor decreases sodium transport by cAMP dependent and cAMP independent mechanisms. Dopamine, via D1-like receptors, may inhibit Na+/H+ exchange activity in renal brush border membranes by a cAMP independent/Gs alpha-linked mechanism. Another cAMP independent pathway of sodium transport inhibition is mediated by phospholipase C, which has several isoforms (PLC beta, PLC gamma, and PLC delta with several members in each). Catecholamines stimulate expression and activity of phospholipase C isoforms in a concentration, time, and receptor-dependent as well as regional and subcellular compartmental-specific manner. In renal cortical membranes, intrarenal administration of norepinephrine for 3-4 h increases PLC beta expression and activity but has no effect on PLC gamma activity. In contrast, intrarenal administration of a D1 agonist for 3-4 h increases PLC beta 1 but decreases PLC gamma expression and activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1995

Robotics and automated workstations for rapid response testing.

Rapid-response testing can help the critical care physician provide more medically relevant decisions when treating critically ill patients. Many technologies have appeared on the market to help deliver rapid analytical tests, including transportation systems, hand-held analyzers, or clinical instruments that have simple user interfaces. Each of these methods can be used to provide the necessary medical information but often at the expense of turnaround time, quality of service, or cost. A robotically automated laboratory system was created that provides rapid turnaround time and low cost, and each result is monitored and reviewed by a laboratory professional. To provide the best quality laboratory services at the lowest cost, we created a remotely controlled robotic clinical laboratory that provides whole-blood analysis of blood gases (pCO2,pO2), pH, electrolytes (sodium, potassium, and chloride), glucose, and hemoglobin near the patient beside yet maintains the distinct advantage of central laboratory control. The automated remote laboratory provides extremely rapid turnaround time, eliminates the costly steps involved with specimen processing, reduces the risk from contaminated specimens, reduces staff training, ensures that every result is reviewed by a professional, and improves patient care.

Automation↗

PubMed1995

Development of a robotic near patient testing laboratory.

Many laboratories have centralized critical care services to conserve resources, but centralization has often been at the expense of providing optimized patient care. We have created a remotely controlled clinical laboratory that provides whole blood analysis of blood gases (PCO2, PO2), pH, electrolytes (Na+, K+, Cl-), glucose, and hemoglobin near the patient's beside, while maintaining the distinct advantage of central laboratory control. The automated remote laboratory provides extremely rapid turnaround times, eliminates the cost of labor for specimen processing, reduces the risk from contaminated specimens, reduces staff training, and results in improved patient care.

Centralized Hospital Services↗

PubMed1994

Role of renal dopamine D1 receptors in natriuresis induced by calcium channel blockers.

The direct tubular natriuretic effect of calcium channel blockers (CCBs) may be due to an interaction between CCBs and a renal tubular dopamine receptor. We therefore studied the effects of two chemically unrelated CCBs, diltiazem and isradipine, infused into the right renal artery of 5% saline-loaded anesthetized rats alone or in the presence of a D1 antagonist, SKF-83742. Isradipine (0.03 microgram.kg-1.min-1) or diltiazem (20 but not 10 micrograms.kg-1.min-1) alone produced an increase in urine flow and an approximate doubling of absolute and fractional sodium excretion, which was not seen in the left kidney or in the control animals (analysis of variance, Scheffé's test, P < 0.05). SKF-83742 alone given systemically or into the right renal artery did not affect these parameters but did block the actions of diltiazem or isradipine. There was no change in mean arterial pressure, renal blood flow, or glomerular filtration rate in any of the experiments. In additional studies, we found that a combined infusion of dopamine (0.1 microgram.kg-1.min-1) and diltiazem (10 micrograms.kg-1.min-1) (doses that by themselves did not alter renal function) produced a twofold or greater increase in urine flow and absolute and fractional sodium excretion; glomerular filtration rate was not significantly changed. Intrarenal arterial CCBs, without a change in renal hemodynamics, produce a natriuresis that is blocked by a D1 antagonist. Concomitant administration of diltiazem and dopamine (each in subeffective doses when used alone) produces a synergistic effect.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Dopaminergic defect in hypertension.

Reverse genetics and the candidate gene approach have been utilized to identify the genetic defect(s) in hypertension. We have proposed the dopamine receptor gene as one candidate in the pathogenesis of hypertension. Because some forms of hypertension are sodium dependent or aggravated by sodium loading and because dopamine is important in aiding the organism to eliminate "excess" sodium, an abnormality in the renal dopaminergic system may be responsible for the sodium retention in hypertension. Both human and animal models of hypertension are associated with renal dopamine production and/or post first messenger defects. The Dahl salt-sensitive rat, which has a decreased ability to generate renal dopamine, and the spontaneously hypertensive rat (SHR), which has no such limitation, have a defective coupling of a D1 receptor to a G protein/adenylyl cyclase complex. This coupling defect is: (1) genetic, since it precedes the onset of hypertension and co-segregates with the hypertensive phenotype, (2) receptor specific, since it is not shared by other humoral agents, and (3) organ and nephron segment selective, since it occurs in proximal tubules but not in cortical collecting ducts or the brain striatum. A consequence of the defective dopamine receptor/adenylyl cyclase coupling in the SHR is a decreased ability of D1 agonists to inhibit Na+/H+ exchange activity. A resistance to the natriuretic effect of dopamine and D1 agonists in the SHR is due mainly to decreased cyclic AMP production, although with maturation a post cyclic AMP defect is acquired. Radioligand binding studies suggest a "loss" of the high-affinity D1 binding site in the SHR.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1993

Organ specificity of the dopamine1 receptor/adenylyl cyclase coupling defect in spontaneously hypertensive rats.

The coupling between the dopamine1 (DA1) receptor and the G protein/adenylyl cyclase (AC) enzyme complex is defective in the proximal convoluted tubule (PCT) of 20-wk-old spontaneously hypertensive rats (SHRs). Because this coupling defect could have been due to desensitization secondary to elevated renal dopamine levels in the adult animal, we studied the interaction between DA1 receptors and AC in PCT of rats as early as 3 wk of age, a time when renal dopamine levels are similar in SHRs and their normotensive controls (Wistar-Kyoto rats, WKYs). Maximum receptor density did not change with age and was similar in WKYs and SHRs in all the age groups studied (3, 8, and 20 wk). Basal-, forskolin-, and guanyl nucleotide-stimulated AC activities were also similar in WKYs and SHRs and did not change with age. However, the DA1 agonist-stimulated AC activity was greater in WKYs than in SHRs and increased with age in WKYs but not in SHRs. Moreover, the ability of a nonhydrolyzable analogue of GTP, Gpp(NH)p, to enhance DA1 agonist (SND-919-C12, 1 microM)-stimulated AC activity increased with age in WKY but not in SHRs. To determine if the defect noted in the PCT of SHRs is due to a defective D1A receptor gene, parallel studies were performed in the striatum, since this receptor is expressed predominantly in the latter tissue. In contrast to the results in PCT, radioligand binding and AC studies in striatum revealed no differences between WKYs and SHRs.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenylyl Cyclases↗

PubMed1993

Nephron specificity of dopamine receptor-adenylyl cyclase defect in spontaneous hypertension.

Since DA1 receptors regulate renal tubular sodium transport, it is possible that the reported defect in the coupling between the DA1 dopamine receptor and adenylyl cyclase (AC) in the proximal tubule (PT) is a mechanism for the increased sodium reabsorption in animal models of spontaneous hypertension. Because the distal nephron may participate in the increased sodium retention in the spontaneously hypertensive rat (SHR), we determined whether the defective DA1 receptor-AC coupling described in PT of SHR is also present in the cortical collecting duct (CCD). Radioligand binding studies with the DA1 antagonist 125I-Sch 23982 revealed similar dissociation constants and maximum receptor densities in the CCD from Wistar-Kyoto rats (WKY) and SHR. Fenoldopam, a DA1-selective agonist, stimulated AC activity to a similar extent in CCD from both rat groups. Therefore the defective DA1 receptor-AC coupling in SHR has nephron segment specificity, since it is present in PT but not in CCD. One of the AC-linked dopamine receptors is an intronless D1A cloned from brain, which is also present in PT. Because the coupling defect in the PT may reside in the third cytoplasmic loop (involved in G protein coupling), we compared the sequence of this segment of the cloned D1A receptor using genomic DNA. Because no differences were noted between WKY and SHR, the coupling defect in the PT is not due to a mutation at the third cytoplasmic loop of the D1A receptor.

Adenylyl Cyclases↗

PubMed1993

Expression of dopamine D1A receptor gene in proximal tubule of rat kidneys.

The existence of dopamine receptor subtypes outside the central nervous system has been suggested by pharmacological and biochemical techniques. Whether the renal dopamine receptors are distinct from those cloned from the brain remains to be determined. Indeed, the expression of any of these receptor genes in the kidney has not been demonstrated definitively. In this study, we amplified D1A receptor cDNA from microdissected proximal convoluted tubules of the rat kidney by reverse transcription-polymerase chain reaction; primers were based on the sequence of rat D1A cDNA cloned from the brain and corresponded to the third cytoplasmic loop of the receptor. Specificity of the amplified products was verified by restriction analysis, Southern blots, and sequencing. Furthermore, solution hybridization indicated the presence of a single ribonuclease-protected RNA species corresponding to the D1A receptor mRNA in proximal tubules. These studies report for the first time the presence of D1A receptor message in the kidney.

Animals↗