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Biomedical subjects

S A Rivkees

Publications and source records attributed to S A Rivkees.

At least 19 recordsLinked to original sources
PubMed1999

Characterization of the murine A1 adenosine receptor promoter, potent regulation by GATA-4 and Nkx2.5.

Adenosine acts via A1 adenosine receptors (A1ARs) in the heart and brain to potently influence mammalian physiology. A1ARs are expressed very early in embryonic development, and A1ARs are among the earliest expressed G protein coupled receptors in the heart and brain. To understand the biologic basis of A1AR expression, a genomic fragment containing the murine A1AR promoter was cloned. Reporter assay studies using DDT1 MF2 cells that express A1ARs revealed that 500 base pairs of the proximal A1AR promoter contained essential elements for A1AR gene expression. Transgenic mice with A1AR proximal promoter coupled with the beta-galactosidase reporter gene had heavy labeling of the brain and atria, consistent with normal patterns of A1AR expression. Within the proximal A1AR promoter, putative binding sites for cardiac transcription factors GATA and Nkx2.5 were identified. Co-expression studies revealed that GATA-4 and Nkx2.5 could individually drive A1AR promoter activity and act synergistically to activate A1AR expression. These observations suggest that embryonic A1AR expression involves activation of the A1AR promoter by GATA-4 and Nkx2.5.

Animals↗
PubMed1999

The biological clock of very premature primate infants is responsive to light.

Each year more than 250,000 infants in the United States are exposed to artificial lighting in hospital nurseries with little consideration given to environmental lighting cycles. Essential in determining whether environmental lighting cycles need to be considered in hospital nurseries is identifying when the infant's endogenous circadian clock becomes responsive to light. Using a non-human primate model of the developing human, we examined when the circadian clock, located in the hypothalamic suprachiasmatic nuclei (SCN), becomes responsive to light. Preterm infant baboons of different ages were exposed to light (5,000 lux) at night, and then changes in SCN metabolic activity and gene expression were assessed. After exposure to bright light at night, robust increases in SCN metabolic activity and gene expression were seen at ages that were equivalent to human infants at 24 weeks after conception. These data provide direct evidence that the biological clock of very premature primate infants is responsive to light.

Animals↗
PubMed1999

Identification of the adenine binding site of the human A1 adenosine receptor.

To provide new insights into ligand-A1 adenosine receptor (A1AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane domains of the human A1AR. First, we replaced eight unique A1AR residues with amino acids present at corresponding transmembrane (TM) positions of A2AARs. We also tested the role of carboxamide amino acids in TMs 1-4, and the roles of Val-87, Leu-88, and Thr-91 in TM3. Following conversion of Gly-14 in TM1 to Thr-14, the affinity for adenosine agonists increased 100-fold, and after Pro-25 in TM1 was converted to Leu-25, the affinity for agonists fell. After conversion of TM3 sites Thr-91 to Ala-91, and Gln-92 to Ala-92, the affinity for N6-substituted agonists was reduced, and binding of ligands without N6 substituents was eliminated. When Leu-88 was converted to Ala-88, the binding of ligands with N6 substituents was reduced to a greater extent than ligands without N6 substituents. Following conversion of Pro-86 to Phe-86, the affinity for N6-substituted agonists was lost, and the affinity for ligands without N6 substitution was reduced. These observations strongly suggest that Thr-91 and Gln-92 in TM3 interact with the adenosine adenine moiety, and Leu-88 and Pro-86 play roles in conferring specificity for A1AR selective compounds. Using computer modeling based on the structure of rhodopsin, a revised model of adenosine-A1AR interactions is proposed with the N6-adenine position oriented toward the top of TM3 and the ribose group interacting with the bottom half of TMs 3 and 7.

Adenine↗
PubMed1999

Home monitoring of 17 hydroxyprogesterone levels in congenitx127drenal hyperplasia with filter paper blood samples.

OBJECTIVE: The purpose of this study was to evaluate the usefulness of 17 hydroxyprogesterone (17OHP) determination in dried filter paper blood samples from patients with congenital adrenal hyperplasia caused by 21-hydroxylase deficiency. It was hypothesized that these home samples would enhance patient treatment. STUDY DESIGN: Results of 17OHP determination in simultaneously collected venous and dried filter paper blood samples were compared to establish assay reliability. Thereafter, parents mailed dried filter paper blood samples collected before each hydrocortisone dose. RESULTS: The 17OHP levels in wet and dried blood samples correlated well (r = 0.98). Results did not change when stored for 2 weeks under various conditions. Blood sampling at different times of the day provided insights into the patterns of 17OHP secretion and identified times of inadequate adrenal suppression. Dose adjustments were then made considering the time of day when adrenal suppression was inadequate. CONCLUSION: Home monitoring of 17OHP is a reliable and practical approach for assessing adrenal steroid activity in patients with congenital adrenal hyperplasia. Considering the time of day of 17OHP elevations also facilitates hydrocortisone dosing adjustment.

17-alpha-Hydroxyprogesterone↗
PubMed1999

Etidronate therapy for hypercalcemia in subcutaneous fat necrosis of the newborn.

Subcutaneous fat necrosis of the newborn (SCFN) is characterized by indurated violet skin nodules and, occasionally, life-threatening hypercalcemia. Current treatments of patients with SCFN-related hypercalcemia are often only partially successful and may be associated with prolonged hypercalcemia. We now report the use of etidronate, a bisphosphonate, to control hypercalcemia in an infant with SCFN.

Etidronic Acid↗
PubMed1998

Evidence for physiologically active axonal adenosine receptors in the rat corpus callosum.

Several neurotransmitter receptors have been identified on axons, and emerging evidence suggests that central axonal conduction may be modulated by neurotransmitters. We have recently demonstrated the presence of extra-synaptic adenosine Al receptors along rat hippocampal axons. We now present immunocytochemical evidence for Al receptors on rat corpus callosum axons and show that these receptors actively modulate axon physiology. Using rat brain coronal slices, we stimulated the corpus callosum and recorded the evoked extracellular compound action potential. The lipid-soluble, Al-specific adenosine receptor agonist cyclopentyladenosine, dose-dependently decreased the compound action potential amplitude, an effect reversed by the specific Al antagonist 8-cyclopentyl-1, 3-dipropylxanthine. These data provide the first direct evidence that axonal Al adenosine receptors modulate axon physiology in the adult mammalian brain. Influencing axonal transmission is a potentially powerful mechanism of altering information processing in the nervous system.

Action Potentials↗
PubMed1998

Localization and functional effects of adenosine A1 receptors on cardiac vagal afferents in adult rats.

There is evidence to suggest that during ischemia adenosine acts on cardiac vagal afferent neurons to activate systemic reflexes and to modulate cardiac nociception. The purpose of this study was to determine whether adenosine receptors are present and have direct cellular electrophysiological actions on cardiac vagal afferent neurons. In radioreceptor assays of nodose ganglion tissue from rats, binding was detectable for A1 (39.6 +/- 1.2 fmol/mg protein) but not A2a adenosine receptors. These findings were confirmed using the complementary approach of receptor-labeling autoradiography. Using in situ hybridization, we saw specific labeling over approximately 50% of neurons in the nodose ganglia, but not over nonneuronal cells. In colabeling studies, cardiac vagal afferent neurons were identified by retroneuronal labeling with fluororuby. Of cardiac vagal afferents approximately one-half were strongly positive for A1 adenosine receptors (immunocytochemistry). In patch-clamping experiments, adenosine inhibited peak inward calcium current in 7 of 11 cells by 48 +/- 13%. In conclusion, adenosine A1 receptors reside on a subset of vagal afferent neurons, including cardiac vagal afferents, and have electrophysiological effects that modulate neuroexcitability in cultured nodose ganglion neurons.

Afferent Pathways↗
PubMed1997

D1 receptors mediate dopamine action in the fetal suprachiasmatic nuclei: studies of mice with targeted deletion of the D1 dopamine receptor gene.

Studies in rodents suggest the presence of a dopaminergic system that influences the function of a biological clock in the hypothalamic suprachiasmatic nuclei (SCN). To provide insights into mechanisms of dopamine action in the SCN, we studied transgenic mice that had either one allele (+¿-) or both alleles (-/-) of the D1 dopamine receptor gene deleted, along with normal (+/+) littermates. As expected, receptor labelling autoradiography studies using [125I]SCH 23982 showed a complete absence of D1 dopamine receptor binding sites in the SCN of -/- animals. When pregnant mice from +¿- x +¿- matings were injected with the D1 receptor agonist SKF 38393, or the dopamine reuptake blocker GBR 12909 at day 19 of gestation, c-fos mRNA expression was observed in the SCN of +/+ fetuses. In contrast, c-fos mRNA induction was not seen in -/- or +¿- litter mates. Injection of cocaine into pregnant dams also resulted in robust SCN c-fos mRNA expression in +/+ mice. Increases in SCN c-fos mRNA expression were also seen in +¿- and -/- mice suggesting that cocaine action in the SCN involves both D1 receptor-dependent and -independent mechanisms. Collectively, our studies of transgenic mice deficient in D1 receptors support the presence of a functional dopaminergic system in the fetal SCN. We also identify D1 receptors as the prominent transducer of dopamine action in the fetal SCN.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗
PubMed1997

Newborn primate infants are entrained by low intensity lighting.

At the present time we do not know when the circadian timing system of human infants becomes responsive to light. Because of human study limitations, it is not currently possible to address this issue in clinical studies. Therefore, to provide insights into when the circadian system of humans becomes responsive to light, baboons were studied. We first assessed if the biological clock located in suprachiasmatic nuclei (SCN) is responsive to light at birth. When term newborn infants were exposed to bright light at night (5000 lux), SCN metabolic activity and c-fos mRNA expression increased, indicating the presence of photic responsiveness. When photic entrainment of developing rhythmicity was examined in infants, low intensity (200 lux) cycled lighting was sufficient to entrain circadian phase. However, low intensity lighting was not sufficient to induce changes in SCN metabolic activity or c-fos mRNA expression. Phase-response studies indicated that light exposure (200 lux) before the onset of activity most effectively shifted circadian phase. These data provide direct evidence that the SCN are responsive to visually mediated light information in a primate at birth. Further consideration of lighting conditions that infants are exposed to is therefore warranted.

Age Factors↗
PubMed1997

Functional D1 and D5 dopamine receptors are expressed in the suprachiasmatic, supraoptic, and paraventricular nuclei of primates.

In rodents, D1 dopamine receptors are expressed in the suprachiasmatic nucleus and are believed to play important roles in regulating circadian rhythms. It is not currently known if the primate circadian system can be influenced by dopaminergic agents, which have broad clinical use. To determine if dopamine receptors can potentially influence primate circadian function, we examined the expression of D1 dopamine receptors in the anterior hypothalamus of ring-tailed macaques (Macaca nemestrema), baboons (Papto sp.), and humans. Because D5 dopamine receptors also stimulate adenylyl cyclase activity, D5 dopamine receptor expression was studied as well. We used [125I]SCH 23982, which binds to D1 and D5 dopamine receptors, and labeling of the suprachiasmatic (SCN), supraoptic (SON), and paraventricular (PVN) nuclei was detectable in each species. In situ hybridization studies revealed differential expression of D1 and D5 dopamine receptor mRNA in the hypothalamus. D1 dopamine receptor mRNA was expressed in the SCN, SON, and PVN. By contrast, D5 dopamine receptor mRNA was expressed only in the SON and PVN of baboons and humans. Injection of the D1/D5 dopamine receptor agonist SKF 38393 at night increased the uptake of 2-deoxy-D-[14C]glucose in the SCN, SON, and PVN of newborn baboons. By contrast, c-fos mRNA expression was induced in the SON and PVN, but not in the SCN. These data show that D1 and D5 dopamine receptors are present in the hypothalamus of primates and show that activation of these receptors acutely influences SCN, SON, and PVN activity.

Animals↗
PubMed1997

Developing circadian rhythmicity. Basic and clinical aspects.

Increasing evidence indicates that the circadian timing system is a fundamental hemostatic system that potently influences human behavior and physiology throughout development. Circadian clock function begins during fetal life, and photic regulation of circadian phase is present at birth in primates. After birth, there is progressive maturation of the circadian system, with day-night rhythms in activity and hormone secretion developing between 1 and 3 months of age. Several disorders of the circadian system are now recognized and include clock disorders and problems related to inadequate entrainment of circadian phase. Treatments for several circadian system disorders are now available and include light therapy and melatonin. With the continued elucidation of circadian system development and influences on human physiology and illness, it is anticipated that consideration of circadian biology will become an increasingly important component of clinical care.

Animals↗
PubMed1997

A1 adenosine receptors potently regulate heart rate in mammalian embryos.

A1 adenosine receptors (A1ARs) have been recently shown to be expressed in rodent embryonic hearts at very early stages of development. To determine the functional significance of fetal cardiac A1AR expression during embryogenesis, murine fetal heart preparations were studied between postconceptual days 9 and 12. Dose-response curves generated using a variety of adenosine agonists revealed that A1AR activation potently regulated fetal heart rates. The A1AR agonist, N6-cyclopentyladenosine, inhibited heart rates in a dose-dependent manner (half-maximal effective concentration = 3.6 x 10(-8) M) and stopped fetal cardiac contractions in 63% of preparations. In contrast, A2a and A2b receptor activation did not alter heart rates, and activation of A3 receptors produced modest declines in heart rates. Endogenous adenosine also acted tonically to suppress fetal heart rates, as demonstrated by the A1AR antagonist 1,3-dipropyl-8-cyclopentylxanthine, increasing heart rates, whereas the adenosine reuptake blocker dipyridamole lowered fetal heart rates. Pertussis toxin treatment blocked A1AR action, showing that A1AR action was G protein mediated. Using drugs that alter cAMP levels and ion channel action, we were able to show that A1AR action involves events mediated by cAMP, ATP-dependent K, L-type calcium, sodium, and chloride channels, and the pacemaker current. These data show that adenosine and A1ARs potently regulate mammalian heart rates via multiple effector systems at very early stages of prenatal development.

Adenosine↗
PubMed1996

Differential expression of the apurinic / apyrimidinic endonuclease (APE/ref-1) multifunctional DNA base excision repair gene during fetal development and in adult rat brain and testis.

The multifunctional mammalian apurinic/apyrimidinic (AP) endonuclease is responsible for the repair of AP sites in DNA. In addition, this enzyme has been shown to function as a redox factor facilitating the DNA binding capability of Jun-Jun homodimers and Fos-Jun heterodimers by altering their redox state and to be involved in calcium mediated transcriptional repression of the parathyroid hormone gene. Previous studies examining the tissue specific distribution of the AP endonuclease (APE) transcript and protein by Northern analysis and enzymatic assays, respectively, have shown that this gene is expressed in all tissues at relatively similar levels. In the current study, adult and fetal rat tissue sections were examined for the expression of the APE transcript in specific subpopulations of cells and during development by in situ hybridization. In the adult brain, the APE transcript showed a widespread, but heterogeneous pattern of expression. Predominant levels of transcript were detected in the suprachiasmatic nuclei, the supraoptic and paraventricular nuclei, the hippocampus and the cerebellum. During fetal development, transcript was detected in all somatic sites examined with very high levels in the thymus, liver and developing brain. Examination of the adult testis indicated that the expression of the transcript varies with the stage of spermatogenesis with the highest levels being present over round spermatids. These results provide evidence that the APE gene is not homogeneously expressed, but rather is found in subpopulations of cells in the brain and testes and during development.

Animals↗
PubMed1996

Site-directed mutagenesis of the human A1 adenosine receptor: influences of acidic and hydroxy residues in the first four transmembrane domains on ligand binding.

To provide new insights into ligand/A1 adenosine receptor (A1 AR) interactions, site-directed mutagenesis was used to test the role of several residues in the first four transmembrane (TM) domains of the human A1 AR. Based on multiple sequence analysis of all known ARs, both acidic (glutamic acid and aspartic acid) and polar hydroxy (serine and threonine) amino acids were identified that could potentially play a role in binding adenosine. Glu16 (TM1), Asp55 (TM2), Ser93 and Ser94 (TM3), Ser135 (TM4), and Thr 141 (TM4) were identified in all ARs, and Ser6 and Ser23 (TM1) were identified in all A1 ARs. To test the role of these residues, each was individually mutated to alanine. When Ala6, Ala23, Ala50, Ala93, Ala135, and Ala141 constructs were tested, affinities for [3H]2-chloro-N6-cyclopentyladenosine (CCPA) and [3H]1,3-dipropyl-8-cyclopentylxanthine (DPCPX) were similar to those seen for the wild-type receptor. After conversion of Glu16 to Ala16, the affinity for [3H]CCPA and other agonists fell 10-100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. After conversion of Asp55 to Ala55, the affinity for [3H]CCPA and other agonists increased < or = 100-fold, whereas the affinity for [3H]DPCPX and other antagonists was not affected. Studies of the Ala55 construct also revealed that Asp55 is responsible for allosteric regulation of binding by sodium because the affinity for [3H]CCPA did not change over broad ranges of sodium concentrations. When Ser94 was converted to Ala94, A1 AR immunoreactivity was present on stable cell lines; however, functional binding sites could not be detected. When Ser94 was converted to Thr94, the affinity for some xanthine antagonists fell. These data show that Glu16 in TM1 and Asp55 in TM2 play important roles in agonist/A1 AR interactions and show that Asp55 is responsible for allosteric regulation of ligand/A1 AR binding by sodium. We also identify Ser94 as an important site for ligand binding.

Allosteric Regulation↗
PubMed1995

Adenosine A1 receptors are located predominantly on axons in the rat hippocampal formation.

The nucleoside adenosine exerts potent biological effects via specific receptors, including the inhibitory A1 adenosine receptor (A1AR). In the hippocampus A1ARs play an important role in regulating neuronal activity. However, the cellular sites of hippocampal A1ARs are undefined. Using in situ hybridization, receptor autoradiography, and single- and double-label immunocytochemistry techniques, we have characterized the cellular sites of A1AR expression in the rat hippocampus. In situ hybridization and receptor autoradiography studies revealed strikingly different patterns of labeling. In situ hybridization studies revealed heaviest labeling of cell bodies in the granular layer of the dentate gyrus and the pyramidal layers of Ammon's horn. In contrast, using [3H]DPCPX, we observed heavy specific labeling over the neuropil in the dentate hilus stratum moleculare, stratum lacunosum-moleculare, stratum radiatum, and stratum oriens, and little labeling over cell bodies. Using single-label immunocytochemistry, A1AR immunoreactivity was found to be heaviest over fibers in regions corresponding with heavy [3H]DPCPX labeling. Double-label florescent confocal microscopy was then used to determine the identity of labeled fibers. A1AR immunoreactivity was found to co-localize with SMI-31 that labels axons, but not with MAP2a,b that labels cell bodies and dendrites, or with synaptophysin that labels synapses. These data identify axons as the predominant site of A1AR expression in hippocampus. Activation of A1ARs may be a powerful mechanism by which adenosine alters axonal transmission to inhibit neurotransmitter release.

Animals↗
PubMed1995

The ontogeny of cardiac and neural A1 adenosine receptor expression in rats.

To provide insights into the sites and mechanisms of adenosine action during fetal life, the ontogeny of A1 adenosine receptor (A1AR) expression was studied in rats. Using in situ hybridization and receptor binding assays, A1AR expression was examined at gestational days (GD) 8, 11, 14, 17, and 21. At GD 8, A1AR mRNA expression was detected in the myocardium but not in other fetal structures. At GD 11, A1AR mRNA was present in the atria, but not in the ventricles or neural structures. At GD 14, A1AR mRNA was present in the atria and the pontine neuroectoderm, the thalamus, and the ventral horn of the spinal cord. At GD 14, A1ARs were first detectable using [3H]DPCPX (8-cyclopentyl-1,3-dipropylxanthine). Functional binding sites were highest in the atria and present at low levels in brain. When GD 17 was reached, patterns of A1AR expression in the brain were similar to those observed in adult animals. Sites of heavy labeling included the spinal cord, medulla, pons, midbrain, thalamus, and the hippocampus. When pre- and postnatal tissue concentrations of A1ARs were compared, cardiac A1AR receptor concentrations were similar. However, postnatal brain A1AR levels were considerably greater than in fetuses. These data identify the atria as a prominent site of fetal A1AR expression and show that the A1AR gene is one of the earliest expressed G protein-coupled receptor genes in the heart. A1AR expression in brain lags behind cardiac expression in early stages of gestation and exceeds cardiac A1AR expression with progressive maturation. A1ARs may therefore influence the heart and brain during critical periods of fetal development.

Animals↗
PubMed1995

Identification of domains of the human A1 adenosine receptor that are important for binding receptor subtype-selective ligands using chimeric A1/A2a adenosine receptors.

To provide new insights into the regions of the human A1 adenosine receptor (A1AR) involved in ligand binding, a series of chimeric human A1 and rat A2a adenosine receptors (A1/A2a) were studied. Binding studies were initially performed on acutely transfected COS cells using fixed doses of the A2aAR agonist [3H]CGS-21680, the A1AR agonist [3H]2-chloro-N6-cyclopentyladenosine (CCPA), and the A1AR antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine (DPCPX). When the region of the A2aAR from the amino terminus to the end of transmembrane (TM) 1 was replaced by the corresponding region of the A1AR (A1TM1/A2a), [3H]CGS-21680 and [3H]CCPA binding was detectable. When an A1TM1-2/A2a construct was studied, [3H]CGS-21680 binding was lost and [3H] DPCPX binding appeared. Saturation studies using [3H]CCPA revealed that the A1TM1/A2a construct had low affinity. However, with the subsequent addition of A1AR TMs 2-4 receptor affinity improved markedly. Saturation studies using [3H]DPCPX also revealed that the TMs 1-4 of the A1AR conferred wild-type receptor affinity. When the ligand binding properties of A1TM1-4/A2a, A1TM1-6/A2a, and wild type A1AR constructs were directly compared, no differences were found using 10 different compounds. When truncated A1ARs that extended from the amino terminus to shortly after TM4 were examined, no binding was detectable suggesting that the amino half of the receptor alone is not sufficient for ligand binding. Collectively, these data suggest that the important determinants for A1AR agonist and antagonist binding and ligand specificity are present in TMs 1-4.

Adenosine↗