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Biomedical subjects

G P Brown

Publications and source records attributed to G P Brown.

At least 19 recordsLinked to original sources

Why do most tropical animals reproduce seasonally? Testing hypotheses on an Australian snake.

Most species reproduce seasonally, even in the tropics where activity occurs year-round. Squamate reptiles provide ideal model organisms to clarify the ultimate (adaptive) reasons for the restriction of reproduction to specific times of year. Females of almost all temperate-zone reptile species produce their eggs or offspring in the warmest time of the year, thereby synchronizing embryogenesis with high ambient temperatures. However, although tropical reptiles are freed from this thermal constraint, most do not reproduce year-round. Seasonal reproduction in tropical reptiles might be driven by biotic factors (e.g., peak periods of predation on eggs or hatchlings, or food for hatchlings) or abiotic factors (e.g., seasonal availability of suitably moist incubation conditions). Keelback snakes (Tropidonophis mairii, Colubridae) in tropical Australia reproduce from April to November, but with a major peak in May-June. Our field studies falsify hypotheses that invoke biotic factors as explanations for this pattern: the timing of nesting does not minimize predation on eggs, nor maximize food availability or survival rates for hatchlings. Instead, our data implicate abiotic factors: female keelbacks nest most intensely soon after the cessation of monsoonal rains when soils are moist enough to sustain optimal embryogenesis (wetter nests produce larger hatchlings, that are more likely to survive) but are unlikely to become waterlogged (which is lethal to eggs). Thus, abiotic factors may favor seasonal reproduction in tropical as well as temperate-zone animals.

Analysis of Variance↗

Expression of Q227L-Galpha(s) inhibits intimal vessel wall hyperplasia after balloon injury.

Interaction between signaling pathways regulates many cellular functions, including proliferation. The Galpha(s)/cAMP pathway is known to inhibit signal flow from receptor tyrosine kinases to mitogen-activated protein kinase (MAPK)-1,2 and, thus, inhibit proliferation. Elevation of cAMP or adenovirus-directed expression of mutant (Q227L)-Galpha(s) (alpha(s)*) inhibits the proliferation of rat vascular smooth muscle cells (VSMCs) in culture. Platelet-derived growth factor (PDGF) stimulated MAPK activation and DNA synthesis was also blocked by expression of alpha(s)*. However, it is not known whether such mechanisms are operative in vivo. Proliferation of vascular smooth muscle cells in vivo was induced by balloon injury of carotid arteries in the rat. Recombinant adenovirus encoding beta-galactosidase (beta-gal) or alpha(s)* was applied to arterial segments injured by the balloon catheters. The alpha(s)*-treated vessels showed decreased phospho-MAPK staining in the intima as compared with beta-gal-treated vessels. Application of alpha(s)*, but not beta-gal containing adenovirus, inhibited formation of neointima by 50%. No change was observed in total vessel diameter or in the media or adventitia. These results suggest that the interaction between the Galpha(s) and MAPK pathways can regulate proliferation in vivo and that targeted expression of activated Galpha(s) may have therapeutic potential for the treatment of vascular pathophysiologies that arise from intimal hyperplasia.

Adenoviridae↗

Long-term potentiation induced by theta frequency stimulation is regulated by a protein phosphatase-1-operated gate.

Long-term potentiation (LTP) can be induced in the Schaffer collateral-->CA1 synapse of hippocampus by stimulation in the theta frequency range (5-12 Hz), an effect that depends on activation of the cAMP pathway. We investigated the mechanisms of the cAMP contribution to this form of LTP in the rat hippocampal slice preparation. theta pulse stimulation (TPS; 150 stimuli at 10 Hz) by itself did not induce LTP, but the addition of either the beta-adrenergic agonist isoproterenol or the cAMP analog 8-bromo-cAMP (8-Br-cAMP) enabled TPS-induced LTP. The isoproterenol effect was blocked by postsynaptic inhibition of cAMP-dependent protein kinase. Several lines of evidence indicated that cAMP enabled LTP by blocking postsynaptic protein phosphatase-1 (PP1). Activators of the cAMP pathway reduced PP1 activity in the CA1 region and increased the active form of inhibitor-1, an endogenous inhibitor of PP1. Postsynaptic injection of activated inhibitor-1 mimicked the LTP-enabling effect of cAMP pathway stimulation. TPS evoked complex spiking when isoproterenol was present. However, complex spiking was not sufficient to enable TPS-induced LTP, which additionally required the inhibition of postsynaptic PP1. PP1 inhibition seems to promote the activation of Ca(2+)/calmodulin-dependent protein kinase (CaMKII), because (1) a CaMKII inhibitor blocked the induction of LTP by TPS paired with either isoproterenol or activated inhibitor-1 and (2) CaMKII in area CA1 was activated by the combination of TPS and 8-Br-cAMP but not by either stimulus alone. These results indicate that the cAMP pathway enables TPS-induced LTP by inhibiting PP1, thereby enhancing Ca(2+)-independent CaMKII activity.

8-Bromo Cyclic Adenosine Monophosphate↗

Thromboxane receptors in human kidney tissues.

Thromboxane (TX) A2 effects in the kidneys include contraction of glomerular mesangial cells and intrarenal vascular tissue. A kidney cDNA encoding a TX receptor expressed in rat renal glomeruli and rat renal arterial smooth muscle cells has been reported. However, TXA2 receptors in human kidneys have not been documented. The purpose of this study was to identify and characterize TXA2 receptors in glomeruli and intrarenal arteries isolated from human kidneys. Normal kidneys, not used for transplant because of technical reasons, were kept at -70 degrees C and used for research purposes. The glomeruli and intrarenal arteries were isolated from renal cortical tissue by a mechanical sieving technique. The equilibrium dissociation constant and receptor number were determined by nonlinear analysis of binding inhibition data. The data were generated in radioreceptor assays using [125I]-BOP, a stable analog of TXA2. The dissociation constants (mean +/- SEM) for binding of I-BOP to human glomeruli and intrarenal arterial membranes were 6.6 +/- 1.1 nM (n = 7) and 20 +/- 6 nM (n = 7), respectively (p < 0.05). The receptor number was 311 +/- 91 fmol/mg protein (n = 7) in glomeruli and 74 +/- 16 fmol/mg protein (n = 7) in intrarenal arterial membranes (p < 0.04). The order of specificity of TXA2 analogs for [125I]-BOP binding sites was similar in glomeruli and in arterial membranes and was I-BOP > or = U46619 > or = pinane TXA2 > or = carbocyclic TXA2 > or = PGH2. These findings provide direct evidence for the presence of specific, high-affinity [125I]-BOP binding sites in human renal glomeruli and extraglomerular vascular tissue. These data also indicate that the human binding sites have higher affinity for the TXA2 agonist I-BOP than for PGH2.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

Gating of CaMKII by cAMP-regulated protein phosphatase activity during LTP.

Long-term potentiation (LTP) at the Schaffer collateral-CA1 synapse involves interacting signaling components, including calcium (Ca2+)/calmodulin-dependent protein kinase II (CaMKII) and cyclic adenosine monophosphate (cAMP) pathways. Postsynaptic injection of thiophosphorylated inhibitor-1 protein, a specific inhibitor of protein phosphatase-1 (PP1), substituted for cAMP pathway activation in LTP. Stimulation that induced LTP triggered cAMP-dependent phosphorylation of endogenous inhibitor-1 and a decrease in PP1 activity. This stimulation also increased phosphorylation of CaMKII at Thr286 and Ca2+-independent CaMKII activity in a cAMP-dependent manner. The blockade of LTP by a CaMKII inhibitor was not overcome by thiophosphorylated inhibitor-1. Thus, the cAMP pathway uses PP1 to gate CaMKII signaling in LTP.

Animals↗

Interaction of gender and dietary protein on renal growth and the renal growth hormone-insulin-like growth factor axis.

The female kidney tends to be smaller, have a lower glomerular filtration rate, and be less susceptible to glomerulosclerosis than the male kidney. Insulin-like growth factor-I (IGF-I) is a peptide growth factor that appears to be important for normal and adaptive kidney growth. The purpose of this study was to compare the kidney growth response of the male and female rat kidneys to increased dietary protein intake and to see whether differences in IGF-I production or receptor expression might underlie any gender differences seen. Male (M) and female (F) Munich-Wistar rats (6 to 9 weeks of age) were randomized to isocaloric diets containing either 20% (NP) or 50% (HP) protein and studied after 3 and 14 days. In the male rat, wet kidney weight was significantly increased with HP at both day 3 (M-HP 1028+/-21 mg vs M-NP 891+/-19 mg, p < 0.01) and day 14 (M-HP 1499+/-41 mg vs M-NP 1246 +/-37 mg, p < 0.01). In contrast in the female rat, while there was evidence of initial increased growth at day 3 in the kidneys of F rats fed HP (F-HP 788+/-39 mg vs F-NP 650+/-23 mg, p < 0.01), this difference was not sustained at 14 days (F-HP 961+/-67 mg vs F-NP 931+/-71 mg, p = NS). At day 3, kidneys of both male and female rats fed HP exhibited an increase in total protein but not DNA content. The kidneys of male rats showed increased protein/DNA ratios in the medulla and inner cortex, whereas in the kidneys of female rats, the increase in protein/DNA ratio was confined to the cortex. After 14 days of HP ingestion, the kidneys of male rats showed increases in total kidney content of both DNA and protein, and protein/DNA ratios returned to control values in whole kidney, inner cortex, and medulla. In contrast, in the kidneys of female rats, not only was overall growth response reduced, but neither total kidney protein content nor DNA content was increased. Increased protein/DNA ratios were seen in inner cortex and in outer and inner medulla, similar to that seen at day 3 in the kidneys of male rats. Neither baseline plasma (M-NP 793+/-10 ng/ml, F-NP 704+/-32 ng/ml, p = NS) nor kidney IGF-I content (M-NP 520+/-55 ng/gm tissue, F-NP 506+/-54 ng/gm tissue, p = NS) differed between male and female rats fed NP diets. Both male and female rats showed a comparable increase in kidney IGF-I after 3 days of HP ingestion, and kidney IGF-I returned to control values by 14 days. There was no significant difference in the number or affinity of glomerular IGF-I receptors between male and female rats. In conclusion, we have shown that in the adult male rat, an increase in dietary protein ingestion results in a sustained increase in kidney size that is initially consistent with a hypertrophic response but subsequently shows elements of hyperplasia. In contrast, in the female rat, although there was evidence of the initial hypertrophic (and IGF-I) responses to increased dietary protein, the increase in kidney size was not sustained. However, these profound gender-based differences in the growth response to dietary protein did not appear to be due to differences in kidney expression of IGF-I or its receptors.

Animals↗

3H-naloxone benzoylhydrazone binding in MOR-1-transfected Chinese hamster ovary cells: evidence for G-protein-dependent antagonist binding.

Naloxone benzoylhydrazone (NalBzoH) is a potent mu antagonist in vivo. In a cell line stably transfected with MOR-1 (CHO/MOR-1), NalBzoH also was an antagonist when examined in adenylyl cyclase studies. In binding studies, it displayed high affinity for the mu receptor, confirming its earlier characterization in brain membranes. In competition studies under equilibrium conditions, NalBzoH and diprenorphine both retained their potency in the presence of the stable GTP analog 5'-guanylylimidophosphate, consistent with their mu antagonist properties, whereas the agonist DAMGO showed more than a 3-fold loss of affinity. The dissociation of 3H-diprenorphine was monophasic. However, kinetic studies revealed biphasic dissociations for both 3H-NalBzoH and 3H-DAMGO. The slow component of 3H-NalBzoH dissociation, corresponding to the higher affinity state, was dependent on coupling to G-proteins. It is selectively abolished by guanine nucleotides, leaving only the rapid dissociation phase. Furthermore, the slow dissociation component is eliminated by treatment of the cells with pertussis toxin, but not cholera toxin. In conclusion, NalBzoH is an unusual opioid. Functionally it is an antagonist, a classification consistent with its equilibrium binding in the presence of guanine nucleotides. Yet, kinetic studies reveal that it labels a G-protein coupled state of the receptor with high affinity.

Adenylate Cyclase Toxin↗

3-Methoxynaltrexone, a selective heroin/morphine-6beta-glucuronide antagonist.

Recent work has suggested that heroin and morphine-6beta-glucuronide (M6G) both act through a novel mu opioid receptor subtype distinct from those mediating morphine's actions. This very high affinity 3H-M6G site is selectively competed by 3-methoxynaltrexone. In vivo, 3-methoxynaltrexone (2.5 ng, i.c.v.) selectively antagonizes the analgesic actions of heroin and M6G without interfering with mu (morphine and [D-Ala2,MePhe4,Gly(ol)5]enkephalin), delta ([D-Pen2,D-Pen5]enkephalin), kappa1 (U50,488H) or kappa3 (naloxone benzoylhydrazone) analgesia. In dose-response studies, 3-methoxynaltrexone (2.5 ng, i.c.v.) significantly shifted the ED50 values for heroin and its active metabolite, 6-acetylmorphine, without affecting the morphine curve. These results indicate that 3-methoxynaltrexone selectively blocks a novel 3H-M6G binding site which is responsible for the analgesic actions of heroin and M6G. This ability to selectively antagonize heroin actions opens new possibilities in the development of therapeutics for the treatment of opioid abuse.

Analgesia↗

Synthesis and characterization of [125I]3'-(-)-iodopentazocine, a selective sigma 1 receptor ligand.

Pentazocine is a potent ligand at both opioid and sigma receptors, but with opposite stereoselectivities. Whereas (-)-pentazocine has high affinity for a number of opioid receptors, (+)-pentazocine labels sigma 1 receptors. Iodination of (-)-pentazocine at the 3'-position reverses its selectivity for opioid and sigma 1 receptors. 3'-(-)-Iodopentazocine competes at sigma 1 receptor binding sites with a Ki value of 8 nM, compared to approximately 40 nM for (-)-pentazocine. 3'-(-)-Iodopentazocine also has lost its affinity for opioid receptors. In contrast, iodination of (+)-pentazocine lowers its affinity at sigma 1 receptors. Synthesis of [125I]3'-(-)-iodopentazocine is readily performed with incorporations of up to 80%. Binding is of high affinity and shows the selectivity anticipated for a sigma 1 receptor-selective ligand. Exposing membranes prebound with [125I]3'-(-)-iodopentazocine to ultraviolet light can covalently couple the ligand into the membranes. Polyacrylamide gel electrophoresis reveals a major band at about 25 kDa and a minor one at about 20 kDa, indicating photolabeling of sigma 1 receptors with minor incorporation into sigma 2 sites.

Affinity Labels↗

3H-morphine-6beta-glucuronide binding in brain membranes and an MOR-1-transfected cell line.

Morphine-6beta-glucuronide (M6G) is a potent morphine metabolite. In an effort to further explore its mechanisms of action, we synthesized 3H-M6G of high specific activity and examined its binding. Although its affinity toward traditional mu receptors is similar to morphine in binding assays in brain and in Chinese hamster ovary cells stably transfected with MOR-1, M6G is >100-fold more potent than morphine in analgesic assays. This apparent discrepancy cannot be explained by differing intrinsic activities of the two drugs because both agents are partial agonists with similar efficacies in adenylyl cyclase assays in the transfected cell lines. Behavioral studies have implied the possibility of a distinct M6G receptor. Detailed binding studies in brain tissue reveal evidence for heterogeneity. Nonlinear regression analysis of 3H-M6G saturation studies reveals two components. The lower-affinity component (K(D) = 1.93 +/- 0.6 nM) corresponds to labeling of traditional mu receptors. In addition, 3H-M6G labels another site of low abundance with very high affinity (K(D) = 68 +/- 7 pM). Competition studies indicate that both sites are relatively mu selective. However, several compounds clearly distinguish between the two sites. These binding studies support the concept of a unique M6G receptor responsible for its analgesic activity.

Animals↗

Novel receptor mechanisms for heroin and morphine-6 beta-glucuronide analgesia.

The rapid metabolism of heroin to 6-acetylmorphine and its slower conversion to morphine has led many to believe that heroin and morphine act through the same receptors and that the differences between them are due to their pharmacokinetics. We now present evidence strongly implying that heroin and two potent mu drugs, fentanyl and etonitazine, act through a unique receptor mechanism similar to morphine-6 beta-glucuronide which is readily distinguished from morphine. Heroin, 6-acetylmorphine and morphine-6 beta-glucuronide show no analgesic cross tolerance to morphine in a daily administration paradigm, implying distinct receptors. Strains also reveal analgesic differences among the drugs. CXBK mice, which are insensitive to morphine, retain their analgesic sensitivity to heroin, 6-acetylmorphine, morphine-6 beta-glucuronide, fentanyl and etonitazine. Antisense mapping of the mu opioid receptor MOR-1 reveals that oligodeoxynucleotide probes against exon 2, which are inactive against morphine analgesia, block morphine-6 beta-glucuronide, heroin, fentanyl and etonitazine analgesia. Finally, an antisense probe targeting Gi alpha 1 blocks both heroin and morphine-6 beta-glucuronide, but not morphine, analgesia. These results indicate that heroin, 6-acetylmorphine, fentanyl and etonitazine all can produce analgesia through a novel mu analgesic system which is similar to that activated by morphine-6 beta-glucuronide.

Analgesics↗

Synthesis and characterization of substituted benzoylhydrazones of naloxone.

Naloxone benzoylhydrazone (NalBzoH) has proved a valuable tool in the investigation of opioid receptor subtypes. In the present study, we have examined a series of derivatives of NalBzoH in which substitutions have been made on the benzoyl ring. Overall, we see dramatic effects on the binding affinities of derivatives against the various opioid receptor subtypes. Although the range of affinities against the mu receptors is quite modest, ranges of the others vary almost 30-fold for kappa 3, 50-fold for kappa 1 and 100-fold for delta and kappa 2 binding. Few substituted derivatives display greater affinity than NalBzoH for any of the receptors, except for delta sites where several derivatives have affinities almost tenfold greater than NalBzoH. Along with the wide variations in affinity, the compounds also appear to exhibited widely divergent activities in traditional bioassays.

Animals↗

Threats against clinicians: a preliminary descriptive classification.

Threats against psychiatrists are common, but existing studies on the subject lack descriptive information about the nature and resolution of the threat. In the present study, clinicians who had received threats were interviewed in person or by telephone, and case histories were summarized. Threats were classified as situational and transferential. Demographic factors, precipitating events, and legal actions taken are described. The manner in which clinicians reacted to threats is also discussed.

Adult↗

Antisense mapping the MOR-1 opioid receptor: evidence for alternative splicing and a novel morphine-6 beta-glucuronide receptor.

Although MOR-1 encodes a mu opioid receptor, its relationship to the pharmacologically defined mu receptor subtypes has been unclear. Antisense mapping now suggests that these subtypes result from alternative splicing of MOR-1. Three oligodeoxynucleotide probes targeting exon 1 and another oligodeoxynucleotide directed against the coding region of exon 4 block supraspinal morphine analgesia, a mu1 action, while five of six oligodeoxynucleotides directed against exons 2 and 3 are inactive. Inhibition of gastrointestinal transit and spinal morphine analgesia, two mu2 actions, are blocked only by the probe against exon 4 and not by those directed against exon 1. In contrast, the analgesic actions of the extraordinarily potent mu drug morphine-6 beta-glucuronide are blocked by six different antisense oligodeoxynucleotides targeting exons 2 and 3, but not by those acting on exons 1 or 4. These results suggest that the mu1 and mu2 receptor subtypes originally defined in binding and pharmacological studies result from alternative splicing of MOR-1 while morphine-6 beta-glucuronide acts through a novel, previously unidentified receptor which is yet another MOR-1 splice variant.

Alternative Splicing↗

Dimensions of dysfunctional attitudes as vulnerabilities to depressive symptoms.

Empirical tests of the diathesis-stress component of A. T. Beck's (1976; A. T. Beck, A. J. Rush, B. F. Shaw, & G. Emery, 1979) cognitive theory of depression have generally not yielded positive results. The resulting focus on conceptual and methodological concerns has diverted attention from the more fundamental issue of how validly vulnerability is measured. The present investigation uses the Dysfunctional Attitude Scale (DAS; A. N. Weissman, 1979; A. N. Weissman & Beck, 1978), the most commonly used measure of vulnerability in this area, but adopts a more in-depth approach by examining DAS factors in addition to the typical total score. This study involved a sample of undergraduates who had never before taken a college-level examination. The dimension of the DAS measured by the Perfectionistic Achievement factor had the strongest relationship to increased depressive symptoms in response to poorer-than-expected performance on the examination. Implications for future research in this area are discussed.

Adolescent↗