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Biomedical subjects

D P Healy

Publications and source records attributed to D P Healy.

At least 19 recordsLinked to original sources
PubMed1992

Effects of intracerebroventricular captopril on angiotensin-converting enzyme and angiotensinogen mRNA levels in rat brain.

The effects of intracerebroventricular (i.c.v.) infusion of the angiotensin-converting enzyme (ACE) inhibitor captopril on angiotensin-induced drinking, brain ACE activity, and ACE and angiotensinogen (A-ogen) mRNA levels were examined. I.c.v. infusion of captopril at a rate of 1 microgram/microliter per h for 7 days resulted in a 60% reduction in brain ACE activity and an 80% reduction in the drinking response to i.c.v. angiotensin I. Quantitative solution hybridization experiments indicated that brain ACE mRNA levels were decreased by 40%, whereas brain A-ogen mRNA levels were unchanged. These results suggest that ACE and A-ogen mRNA levels are regulated differently in the brain than in the peripheral renin-angiotensin system.

Angiotensinogen↗
PubMed1992

Characterization of an angiotensin type-1 receptor partial cDNA from rat kidney: evidence for a novel AT1B receptor subtype.

We sought to determine if multiple forms of mRNA for the angiotensin type-1 (AT1) receptor could be detected in rat kidney using the polymerase chain reaction (PCR) procedure. Amplification of rat kidney cDNA with oligonucleotide primers derived from the second and sixth transmembrane domains of the rat AT1 receptor yielded a single cDNA fragment 528bp in size. Sequence analysis indicated, however, that the cDNA fragment was a mixture of two highly similar gene products: the first cDNA was identical to the previously cloned AT1 receptor (termed here AT1A) whereas the second cDNA (termed here AT1B) was 92% identical at the nucleotide level and 96% identical at the amino acid level. Nucleotide substitutions were dispersed throughout the cDNA and 80% (33 of 41) were conservative. Significant levels of AT1A and AT1B mRNA were detected by PCR amplification of kidney poly(A)+ RNA and restriction enzyme analysis. These results indicate that at least two distinct AT1 receptor genes are expressed in rat kidney.

Amino Acid Sequence↗
PubMed1992

Immunocytochemical localization of endopeptidase 24.15 in rat brain.

Endopeptidase 24.15 (EC 3.4.24.15; EP 24.15), a zinc-metalloendopeptidase highly active in rat testes, brain and pituitary, converts some prodynorphin- and proenkephalin-derived oligopeptides into the corresponding enkephalins and degrades a variety of bioactive peptides including bradykinin, neurotensin, and both angiotensin I and II. The immunocytochemical localization of the enzyme was studied in rat brain using a polyclonal antibody raised in rabbits against a homogeneous preparation of the enzyme isolated from rat testes. The distribution of EP 24.15 immunoreactivity in the brain was widespread, being present in both neurons and glial cells. Surprisingly, however, staining was predominantly nuclear, and not cytoplasmic as expected based on the biochemical demonstration that EP 24.15 activity is predominantly associated with the soluble protein fraction of brain homogenates. Cytoplasmic staining was detected in large neurons but was less intense than the nuclear staining. The highest density of EP 24.15-staining was detected in nuclei of cerebellar Purkinje cells and in hippocampal dentate gyrus cells. High levels of immunoreactivity were also noted in brain areas which contain peptides known to be substrates of the enzyme in vitro. This localization supports a role for EP 24.15 in neuropeptide metabolism, but also suggests an as yet undefined role in nuclear function.

Animals↗
PubMed1992

A68930 is a potent, full agonist at dopamine1 (D1) receptors in renal epithelial LLC-PK1 cells.

The selective dopamine1 (D1) receptor agonists SK&F 82526 (fenoldopam) and A68930 and the mixed D1/D2 agonist SK&F 85174 were tested for their ability to stimulate adenosine 3':5'-cyclic monophosphate (cyclic AMP) accumulation in the porcine renal epithelial cell line, LLC-PK1. SK&F 82526 and SK&F 85174 were potent stimulators of cyclic AMP accumulation (EC50s 21.4 and 14.5 nM, respectively), but only partial agonists (intrinsic activities 31% and 46% of dopamine respectively). In contrast, A68930 was a potent, full agonist (EC50 12.7 nM, intrinsic activity 102% of dopamine). The stimulatory effects of A68930 and dopamine on cyclic AMP accumulation were not additive, and the stimulation of cyclic AMP accumulation by A68930 was blocked by the D1-selective antagonist, SCH 23390. These properties of A68930 suggest that it may be a useful D1-selective agonist to study renal D1 receptor mechanisms in vitro and in vivo.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1992

Stimulation of angiotensinogen mRNA levels in rat pituitary by estradiol.

Angiotensin II (ANG II) is a putative paracrine hormone in the anterior pituitary. Angiotensinogen mRNA, however, is not detectable by Northern blot hybridization, suggesting that ANG II may not be synthesized within the pituitary. An alternative explanation may be that angiotensinogen gene activity is low under normal conditions, with angiotensinogen mRNA being below the level of detection. Utilizing a sensitive solution hybridization method, we sought to determine whether angiotensinogen mRNA could be detected in pituitaries from normal male rats or ovariectomized (OVX) rats treated with estradiol (E2) for 4 days. Very low levels of angiotensinogen mRNA were detected from male or OVX rat pituitaries, but E2 treatment resulted in a marked dose-dependent increase in pituitary angiotensinogen mRNA levels. Levels of angiotensinogen within the pituitary were not significantly different after the E2 treatment. Angiotensinogen mRNA levels in liver and brain were much higher than in the pituitary but were not altered significantly by the chronic E2 treatment. These results are consistent with the local synthesis of angiotensinogen in the pituitary and further suggest that pituitary angiotensinogen gene transcription is regulated by estrogen.

Angiotensinogen↗
PubMed1992

Angiotensin II receptors in the solitary-vagal area of hypertensive rats.

Angiotensin II (Ang II) has been proposed to be an endogenous neuromodulator of the baroreceptor reflex at the level of the brain stem solitary-vagal area. Elevated activity of the brain Ang II system has been implicated in the development and maintenance of hypertension in spontaneously hypertensive rats and deoxycorticosterone acetate-salt hypertensive rats. In the present study, we sought to determine if Ang II receptors in the solitary-vagal area exhibited altered binding kinetics in spontaneously hypertensive rats or deoxycorticosterone-salt hypertensive rats. Ang II receptors were examined by quantitative autoradiographic analysis of iodine-125-labeled [Sar1,Ile8]Ang II binding in the solitary-vagal area in six groups of animals: 1) spontaneously hypertensive rats, 2) normotensive Wistar-Kyoto rats, 3) uninephrectomized rats, 4) uninephrectomized rats with a 1% solution of saline for drinking water, 5) uninephrectomized and deoxycorticosterone-treated rats, and 6) uninephrectomized and deoxycorticosterone-treated rats given a 1% solution of saline for drinking water. Blood pressure was significantly elevated in the spontaneously hypertensive rats and deoxycorticosterone-salt rats relative to control animals. There was a significant decrease in the binding affinity (increased KD) for 125I-[Sar1,Ile8]Ang II and a significant increase in the maximum binding density for 125I-[Sar1,Ile8]Ang II in the solitary-vagal area of spontaneously hypertensive rats relative to Wistar-Kyoto rats. Deoxycorticosterone-salt rats also exhibited significantly higher KD and maximum binding density values compared with controls. These results indicate that Ang II receptor binding is altered in the solitary-vagal area of two different models of experimental hypertension and suggest that these changes could contribute to the expression of the hypertensive state.

Angiotensin II↗
PubMed1992

In vitro delivery of crushed ciprofloxacin through a feeding tube.

OBJECTIVE: The purpose of this study was to determine the relative delivery of ciprofloxacin through a small-bore feeding tube (8 fr, 106 cm, Argyle). DESIGN: Ciprofloxacin 750-mg tablets were individually ground in a mortar and dissolved in 50 mL of water. Each resulting solution was drawn into a syringe and injected into a glass beaker (n = 10) or through a feeding tube into a beaker (n = 10) over one minute. Syringe apparati were flushed with two 60-mL portions of water. Aliquots from each of the 20 beakers were taken in triplicate, diluted 1:1000 with water, and assayed by HPLC. RESULTS: Ciprofloxacin concentrations (mean +/- SD) for the samples without and with feeding tubes were 11.77 +/- 0.78 mumol/L and 12.16 +/- 0.65 mumol/L, respectively. The 3.3 percent difference is statistically significant (p = 0.04), but clearly is not clinically significant. CONCLUSIONS: Administration of crushed ciprofloxacin through a small-bore feeding tube in the manner described does not result in any measurable loss of drug delivered to the gastrointestinal tract.

Chromatography, High Pressure Liquid↗
PubMed1992

Radioimmunocytochemical localization of corticotropin-releasing factor and adrenocorticotropin in the hypothalamo-hypophyseal system of the rat: effects of adrenalectomy.

Various radioimmunocytochemical approaches have been utilized to localize primary antibody-antigen complexes. Here we examined the binding properties of three different radioiodinated compounds for their ability to label the antibody-antigen complex, including: donkey anti-rabbit immunoglobulin, donkey anti-rabbit F(ab')2-IgG, and a biotinylated goat anti-rabbit secondary antibody followed by [125I]-avidin. These probes were used to localize rabbit primary antisera against corticotropin-releasing factor (CRF) and adrenocorticotropin-releasing hormone (ACTH) in the hypothalamo-hypophyseal system of the rat. The pattern of labeling with each radiolabeled probe was consistent with the light microscopic immunocytochemical staining for CRF and ACTH. The utility of the radioimmunocytochemical method for quantitative analyses was further tested by studying the effects of adrenalectomy (ADX) on the levels of immunoreactive CRF and ACTH in the hypothalamo-hypophyseal system. Computer-assisted microdensitometric analysis of immunoreactive CRF levels in the median eminence indicated that there was a 33% decrease 24 h after ADX. Immunoreactive ACTH levels in the anterior pituitary were significantly decreased from 1 day (38%) to 1 week (36%) after ADX and were increased at 2 weeks (89%). The changes in CRF and ACTH levels, as measured radioimmunocytochemically after ADX, were consistent with previous biochemical studies. These results indicate that computer-assisted radioimmunocytochemical analysis can be used quantitatively to measure immunoreactivity in tissue sections. The high resolution and high sensitivity provided by this method should make it widely applicable.

Adrenalectomy↗
PubMed1991

Fenoldopam is a partial agonist at dopamine-1 (DA1) receptors in LLC-PK1 cells.

Fenoldopam [6-chloro-7,8-dihydroxy-1-(4'-hydroxyphenyl)-2, 3,4,5-tetrahydro-(1H)-3-benzazepine] is a selective dopamine-1 (DA1) agonist with natriuretic/diuretic properties. A component of the natriuretic response to fenoldopam may involve direct DA1 receptor-mediated effects on proximal tubule sodium reabsorption, possibly through stimulation of adenylyl cyclase. Here, we compared the effects of fenoldopam and DA in stimulating cyclic AMP (cAMP) synthesis in LLC-PK1 cells, a renal epithelial cell line that has proximal tubule-like properties and expresses a DA1 receptor linked to stimulation of adenylyl cyclase. Fenoldopam stimulated cAMP accumulation in LLC-PK1 cells in a dose-dependent manner, an effect which could be blocked by the DA1-selective antagonist Sch 23390. Although fenoldopam was more potent than DA (EC50 55.5 +/- 7.75 nM vs. 1.65 +/- 0.64 microM) in stimulating cAMP accumulation in LLC-PK1 cells, the maximum stimulation obtained by fenoldopam was only 37% of the maximum stimulation obtained by DA(Emax 13.0 +/- 2.95 pmol/mg of protein vs. 35.6 +/- 10.19 pmol/mg of protein). Simultaneous incubation of DA and fenoldopam resulted in lower cAMP levels than with DA alone. Incubation of DA with increasing concentrations of fenoldopam produced parallel rightward shifts in the DA dose-response curves. Schild analysis further indicated that fenoldopam acted as a competitive antagonist in the presence of DA, with a pA2 value of 7.38 and a slope of unity. These results indicate that fenoldopam is a partial agonist with low efficacy at DA1 receptors linked to cAMP generation in the LLC-PK1 cells.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1991

Characterization of a dopamine receptor (DA2K) in the kidney inner medulla.

Dopamine (DA) produces a natriuretic/diuretic response in the kidney by mechanisms that are still not well understood. There is some indication that DA2 receptors may be involved in mediating the effects of DA, but little is known regarding the nature of this receptor in the kidney. Autoradiographic localization of [3H]spiperone, a DA2 antagonist, indicated that high-density binding was restricted to inner medullary collecting ducts (IMCDs). [3H]Spiperone binding was saturable, high affinity (Kd, 17.2 +/- 1.65 nM), and high density (Bmax, 935 +/- 83 fmol per mg of protein). The photosensitive spiperone analogue N-(p-azido-m-[125I]iodophenethyl)spiperone labeled similar sized proteins of Mr = 120,000 in membranes prepared from the kidney inner medulla, striatum, and pituitary. However, the rank-order competition profile for the [3H]spiperone binding in the kidney inner medulla differed from the DA2 receptor in striatum and pituitary and, furthermore, RNA (Northern) blot analyses of kidney inner medullary RNA with brain DA2 receptor oligonucleotide probes were negative. Functionally, DA stimulated prostaglandin E2 production by IMCD cells, an effect that could be blocked by the DA2 antagonist domperidone. These results indicate that the kidney inner medulla expresses a functional DA receptor that may represent a newly identified DA receptor subtype (here designated DA2K). Moreover, these results suggest that the kidney inner medulla may be a significant site at which DA, either directly or indirectly, influences water and electrolyte excretion.

Animals↗
PubMed1991

Lack of interaction between lomefloxacin and caffeine in normal volunteers.

Sixteen healthy, nonsmoking adult males participated in a randomized, double-blind, placebo-controlled, two-way crossover study to evaluate the influence of chronic lomefloxacin administration on the disposition of caffeine and its major metabolite, paraxanthine, at steady-state conditions. Lomefloxacin (400 mg) or placebo was administered orally once daily for 5 days to xanthine-free volunteers after an overnight fast. Caffeine (200 mg orally) was administered simultaneously with lomefloxacin on days 3 through 5. After a 2-day washout period, subjects were crossed over to the alternate 5-day regimen with caffeine, which was again given on the final 3 days. Blood samples for caffeine, paraxanthine, and lomefloxacin concentration determinations were serially collected for 48 h following the last dose of each regimen. All compounds were analyzed by high-performance liquid chromatography. For the placebo versus lomefloxacin-containing treatments, maximum caffeine concentrations in plasma (4.35 +/- 0.63 versus 4.07 +/- 0.56 micrograms/ml), areas under the concentration-time curve from time zero to 24 h at steady state (30.3 +/- 6.9 versus 29.7 +/- 6.6 micrograms.h/ml), and elimination half-lives of caffeine (4.8 +/- 1.1 versus 4.8 +/- 1.2 h) were not significantly different. In addition, there were no significant changes in the disposition parameters of paraxanthine as a result of lomefloxacin administration. The frequencies of central nervous system-related effects for the two treatments were not statistically different. We conclude that lomefloxacin has no significant effect on the disposition of caffeine in young healthy volunteers.

4-Quinolones↗
PubMed1991

Locally formed dopamine stimulates cAMP accumulation in LLC-PK1 cells via a DA1 dopamine receptor.

Proximal tubules have been shown to produce dopamine (DA) from (-)-3-(3,4-dihydroxyphenyl)-L-alanine (L-dopa) and to express DA1 dopamine (DA1) receptors linked to inhibition of sodium transport. The LLC-PK1 renal epithelial cell line expresses proximal tubule cell-like properties in vitro. Here, we sought to determine whether the LLC-PK1 cell line would be a useful model system to study dopaminergic mechanisms in vitro. LLC-PK1 cells contained high levels of aromatic L-amino acid decarboxylase (AADC) (Km 0.19 +/- 0.08 mM, Vmax 3.69 +/- 0.57 nmol.mg-1.min-1) and converted L-dopa to DA in a nonsaturable fashion up to 1 mM L-dopa. DA production was blocked by the AADC inhibitor carbidopa. Dopamine stimulated adenosine 3',5'-cyclic monophosphate (cAMP) accumulation in LLC-PK1 cells in a dose-dependent manner (50% effective concentration, 1.53 +/- 0.38 microM; maximal stimulation, 46.6 +/- 10.88 pmol/mg protein); this effect was blocked by addition of DA1-receptor antagonists. L-Dopa also stimulated cAMP accumulation, and this effect was attenuated by an equimolar concentration of carbidopa and blocked by the DA1 antagonist Sch 23390. These results indicate that LLC-PK1 cells convert L-dopa to DA, which then stimulates cAMP via a DA1 receptor coupled to activation of adenylate cyclase. Moreover, the demonstration that locally formed DA can act as an autocrine/paracrine substance in LLC-PK1 cells in vitro is consistent with a role for DA as an autocrine/paracrine substance in vivo.

Animals↗
PubMed1991

Prostaglandin E2 production in rat IMCD cells. I. Stimulation by dopamine.

Previous studies from our laboratory have determined that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to prostaglandin E2 (PGE2) production. In the present study, we have further characterized the dopamine-stimulated PGE2 response. Dopamine stimulated PGE2 production in cultured IMCD cells dose dependently (concentration for half-maximal stimulation, 11.1 microM; maximal stimulation, 235.1% of basal), an effect that was blocked by the DA2 antagonists domperidone and (S)-(-)-3-iodo-2-hydroxy-6-methoxy-N-[(1-ethyl-2-pyrrolidinyl)-methyl] benzamine. Inhibition of intracellular calcium release with 8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate hydrochloride (100 microM) blocked the dopamine response, whereas voltage-dependent calcium-channel blockers had no effect. Inhibition of phospholipase A2 (PLA2) activity with quinacrine (100 microM) completely blocked the dopamine-stimulated PGE2 production, whereas inhibition of polyphosphoinositol hydrolysis with neomycin (100 microM) or inhibition of protein kinase C with 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (10 microM) did not. Pertussis toxin (PT) treatment completely blocked the dopamine-stimulated PGE2 production but not the arachidonic acid-stimulated PGE2 production. These results suggest that dopamine, acting through the DA2K receptor, may be an important regulator of PGE2 production in IMCD cells. Furthermore, our results are most consistent with either a direct interaction of the DA2K receptor with PLA2 through a PT-sensitive G protein or an indirect interaction with PLA2 through mobilization of intracellular calcium.

Animals↗
PubMed1991

Prostaglandin E2 production in rat IMCD cells. II. Possible role for locally formed dopamine.

Dopamine has been proposed as an intrarenal natriuretic hormone. We reported previously that inner medullary collecting duct (IMCD) cells express a novel DA2-like dopamine receptor (namely, DA2K) that is linked to stimulation of prostaglandin E2 (PGE2) production. In this study we examined whether locally formed dopamine could stimulate PGE2 production in cultured IMCD cells. L-Dopa stimulated PGE2 production dose dependently in cultured IMCD cells (concentration for half-maximal stimulation, 54.3 microM; maximal stimulation, 212.7% of basal), with the maximal stimulation similar to that obtained with dopamine. This effect was blocked by aromatic L-amino acid decarboxylase (AADC) inhibitors and DA2-receptor antagonists. IMCD cells also had measurable AADC activity and produced dopamine from exogenously added L-dopa. AADC inhibitors and DA2 antagonists also lowered basal PGE2 levels, suggesting that dopamine was being formed constitutively in culture. These results suggest that cultured IMCD cells have the capacity to take up and convert L-dopa to dopamine, which then stimulates PGE2 production via DA2K receptors. These results further suggest that locally formed dopamine could act as an autocrine/paracrine hormone in the kidney inner medulla to regulate PGE2 synthesis and water and electrolyte excretion.

Animals↗
PubMed1991

[3H]domperidone binding to the kidney inner medullary collecting duct dopamine-2K (DA2K) receptor.

Previous studies by our laboratory have indicated that inner medullary collecting ducts (IMCDs) express a novel dopamine (DA) receptor, designated DA2K, that is linked to stimulation of prostaglandin E2 production. This receptor has a distinct pharmacological profile and is similar in size, but not homologous to, the brain D2 receptor. Because the DA2-selective antagonist domperidone blocks DA-mediated stimulation of prostaglandin E2 production in IMCD cells, we utilized [3H]domperidone to study the binding characteristics of the DA2K receptor in IMCD cells. [3H]Domperidone binding was saturable and best fit to a single high density site (KD, 57.6 +/- 10.5 nM; Bmax, 14.9 +/- 2.7 pmol/mg protein). The specificity of [3H]domperidone binding in IMCD cells was verified by competition analysis with a variety of dopaminergic and nondopaminergic agents. Dopaminergic drugs were less potent competitors for [3H]domperidone binding in IMCD cells than previously reported for brain DA receptors, but the rank order was consistent with the labeling of a DA receptor [antagonists: domperidone greater than spiperone greater than haloperidol greater than Sch 23390 much greater than (-)-sulpiride; agonists: norapomorphine greater than fenoldopam much greater than dopamine = quinpirole], and was better correlated with the pharmacological profile for the brain D2 receptor than the brain D3 receptor. In addition, quinpirole, the most D3-selective ligand currently available, did not compete for [3H]domperidone binding in IMCD cells. These results add further support to the existence of a novel high density DA receptor, DA2K, expressed in IMCD cells.

Animals↗
PubMed1990

Vancomycin-induced histamine release and "red man syndrome": comparison of 1- and 2-hour infusions.

Vancomycin-induced "red man syndrome" (RMS) is mediated in part by histamine release, and its severity is correlated with the area under the plasma histamine concentration-time curve. Ten adult male volunteers participated in a randomized, double-blind, two-way crossover trial (1-week washout interval between regimens) to determine the effect of 1- and 2-h infusions of vancomycin (1.0 g) on histamine release and on the frequency and severity of RMS. The severity of RMS was classified a priori as mild, moderate, or severe from a combined score of pruritus and extent of erythema. Serial concentrations of histamine in plasma and concentrations of vancomycin in serum were measured at baseline and during and after each infusion. Of 10 subjects, 8 had evidence of RMS during the 1-h infusion (3 mild, 3 moderate, and 2 severe), whereas only 3 of the 10 subjects (all mild) had RMS during the 2-h infusion (P less than 0.05). The 1-h infusion was associated with a significantly greater peak concentration of histamine in plasma (1.8 +/- 0.7 versus 1.0 +/- 0.3 ng/ml, P = 0.004) and a greater total release of histamine (74.3 +/- 54.1 versus 36.4 +/- 22.6 ng.min/ml, P = 0.017) than was the 2-h infusion. These data suggest that administration of vancomycin over 2 h reduces the frequency and severity of RMS and the amount of histamine released compared with those after a 1-h infusion in healthy volunteers.

Adult↗
PubMed1990

Comparison of vancomycin- and teicoplanin-induced histamine release and "red man syndrome".

Twelve healthy adult males participated in a double-blind, randomized, two-way crossover study to determine histamine release and the frequency and severity of "red man syndrome" (RMS) following intravenous administration of vancomycin (15 mg/kg of body weight over 60 min) and teicoplanin (15 mg/kg over 30 min). Concentrations of vancomycin and teicoplanin in serum and concentrations of histamine in plasma were measured at baseline and during and after each infusion. Erythema and pruritus were classified a priori as mild, moderate, or severe. The extent of erythema was determined by the use of a burn chart, and pruritus was assessed by the subject with a rank scale. Global severity of RMS was determined by summation of the individual scores for pruritus and erythema. Baseline areas under the concentration-time curve for histamine were not significantly different for the vancomycin and teicoplanin treatments. Vancomycin caused RMS in 11 of 12 subjects (9 severe and 2 moderate cases) and was associated with a significant increase in plasma histamine (46.7 +/- 31.3 ng.min/ml, P less than 0.05). In contrast, teicoplanin did not cause RMS or elicit significant histamine release (8.7 +/- 13.2 ng.min/ml). Peak concentrations of vancomycin and teicoplanin in serum were 58.8 +/- 8.4 and 148.0 +/- 31.8 micrograms/ml, respectively (P less than 0.05). Assuming equal efficacy, these data suggest that teicoplanin may be a safe alternative agent in subjects experiencing severe RMS due to vancomycin; however, further studies in the clinical setting are needed.

Adult↗