Monoclonal antibodies to CD44 and their influence on hyaluronan recognition.

Antibodies to CD44 have been used to inhibit a variety of processes which include lymphohemopoiesis, lymphocyte migration, and tumor metastasis. Some, but not all, CD44-mediated functions derive from its ability to serve as a receptor for hyaluronan (HA). However, sites on CD44 that interact with either ligands or antibodies are poorly understood. Interspecies rat/mouse CD44 chimeras were used to analyze the specificity of 25 mAbs and to determine that they recognize at least seven epitopes. Amino acid substitutions that resulted in loss of antibody recognition were all located in the region of homology to other cartilage link family proteins. While at least five epitopes were eliminated by single amino acid replacements, multiple residues had to be changed to destroy binding by other antibodies. One antibody was sensitive to changes in any of three separate parts of the molecule and some antibodies to distinct epitopes cross-blocked each other. Certain antibodies had the ability to increase HA binding by lymphocytes but this did not correlate absolutely with antibody specificity and was only partially attributable to CD44 cross-linking. Antibodies that consistently blocked HA recognition were all sensitive to amino acid changes within a short stretch of CD44. Such blocking antibodies interacted with CD44 more strongly than ligand in competition experiments. One large group of antibodies blocked ligand binding, but only with a particular cell line. This detailed analysis adds to our understanding of functional domains within CD44 and requirements for antibodies to influence recognition of one ligand.

1-(m-chlorophenyl)piperazine (mCPP) interactions with neurotransmitter receptors in the human brain.

The affinity of 1-(m-chlorophenyl)piperazine (mCPP) for 11 neurotransmitter receptor binding sites was determined in human brain membranes. mCPP is essentially equipotent at all 5-hydroxytryptamine (5-HT) receptor subtypes (IC50 values ranging from 360 to 1300 nM). The drug displays similar affinity (IC50 = 570 nM for alpha 2-adrenergic receptors labeled for 3H-rauwolscine. mCPP is less potent at alpha 1- and beta-adrenergic, dopamine, and muscarinic cholinergic receptors (IC50 values 2500-24,000 nM). mCPP is inactive at both benzodiazepine receptors and the 5-HT uptake sites at concentrations below 100,000 nM. These data demonstrate that mCPP displays similar potency for multiple neurotransmitter receptor binding sites in human brain.

Analysis of tandospirone (SM-3997) interactions with neurotransmitter receptor binding sites.

The interactions of tandospirone (formerly called SM-3997) with 5-HT and other neurotransmitter receptor binding sites were determined in brain homogenates. Tandospirone is most potent at the 5-HT1A receptor, displaying a Ki value of 27 +/- 5 nM. The agent is approximately two to three orders of magnitude less potent at 5-HT2, 5-HT1C, alpha 1-adrenergic, alpha 2-adrenergic, and dopamine D1 and D2 receptors (Ki values ranging from 1300 to 41000 nM). Tandospirone is essentially inactive at 5-HT1B receptors; 5-HT uptake sites; beta-adrenergic, muscarinic cholinergic, and benzodiazepine receptors. This pharmacological profile differs slightly from that of other novel anxiolytics such as buspirone, ipsapirone, and gepirone. Saturation and competition studies using 3H-tandospirone also suggest that the drug interacts with 5-HT1A receptor binding sites in rat cortical membranes (KD = 4.5 +/- 0.8 nM; Bmax = 2.2 +/- 0.6 pmol/g tissue). Based on adenylate cyclase studies which measure 5-HT1A receptor-mediated effects, tandospirone displays approximately 60% of the agonist effect of 8-OH-DPAT, a selective 5-HT1A agonist. Thus, the primary pharmacological effect of tandospirone appears to be partial agonism at the 5-HT1A receptor, an activity similar to other pyrimidinyl-piperazines which are being developed as novel anxiolytic agents.

Differential management of cardiovascular disease in ESRD by nephrologists and cardiologists.

BACKGROUND: Mortality and morbidity from cardiovascular disease are high in patients with end-stage renal disease (ESRD). The cardiovascular profile of patients with ESRD may differ from that of the general population. We examined how nephrologists and cardiologists differ in managing hypertension and cardiovascular disease in patients with ESRD. METHODS: Seven cases incorporating relevant clinical and echocardiographic findings common to this population were developed based on a chart review of an ESRD population. Each ESRD case incorporated a clinical presentation designed to test for a specific dichotomous response to a common and important clinical problem. Nine nephrology and 7 cardiology faculty members, each paired with a senior clinical fellow from the discipline, were surveyed. RESULTS: Nephrologists were less likely than cardiologists to initiate beta-blockade in patients with echocardiographic evidence of regional wall motion abnormalities if there was no history of coronary artery disease (CAD; P < 0.01). In patients with known CAD, cardiologists were more likely than nephrologists to intensify antihypertensive therapy, even in the setting of a history of fractures associated with orthostatic hypotension (P < 0.02). Decision making did not differ between subspecialists in the management of left ventricular hypertrophy, congestive heart failure, or diastolic dysfunction. CONCLUSION: Nephrologists and cardiologists differ in their management of hypertension in the presence of ischemic heart disease in the ESRD population. Only limited data specific to the ESRD population are available to assess which approach is superior. The discipline-driven differential management approaches observed emphasize the need for better evidence-based management strategies for this population.

Differential interactions of traditional and novel antiemetics with dopamine D2 and 5-hydroxytryptamine3 receptors.

The affinities of 11 drugs for both dopamine D2 and 5-hydroxytryptamine3 (5-HT3) receptor sites were determined in brain membranes. The five "traditional" antiemetics (chlorpromazine, prochlorperazine, droperidol, fluphenazine, and domperidone) displayed high affinity (less than 20 nM) for dopamine D2 receptors in corpus striatum but were inactive at 5-HT3 receptors. In contrast, five recently developed 5-HT3 antagonists (BRL 43694, ICS 205-930, zacopride, Lilly 278584, and MDL 72222) displayed nanomolar affinity for the 5-HT3 site but were inactive (greater than 10,000 nM) at the dopamine D2 receptor. Metoclopramide was unique among these agents in that it was similarly potent at dopamine D2 (240 +/- 60 nM) and 5-HT3 (120 +/- 30 nM) receptors.

Identification of 5-hydroxytryptamine1D binding sites in human brain membranes.

High-affinity, specific 3H-5-hydroxytryptamine (5-HT) binding was analyzed in membrane homogenates of human frontal cortex, caudate, and globus pallidus. 5-HT1A and 5-HT1C binding sites were pharmacologically blocked using 100 nM 8-hydroxy-N,N-dipropyl-2-aminotetralin (8-OH-DPAT) and 100 nM mesulergine, respectively. The majority of 5-HT1 sites remained in each of the three brain regions under these conditions. The pattern of nucleotide interactions with these binding sites (GppNHp = GTP = GDP greater than GMP = adenine nucleotides) suggests a possible linkage to a G protein. RU 24969 competition studies confirmed the absence of 5-HT1B binding sites in human cortex, caudate, and globus pallidus. Drug interactions with putative 5-HT1D binding sites in bovine caudate membranes correlated significantly with their affinities for human membrane recognition sites labeled by 3H-5-HT in the presence of 100 nM 8-OH-DPAT + 100 nM mesulergine. We conclude that the majority of 3H-5-HT labeled recognition sites in human cortex, caudate, and globus pallidus represent 5-HT1D binding sites.

Nifedipine versus propranolol for the initial prophylaxis of migraine.

We conducted a randomized open-labeled study of nifedipine versus propranolol for the initial prophylaxis of migraine. Propranolol was effective in 67% of patients (12/18) and well tolerated. Nifedipine was effective in only 30% of patients (6/20). The lack of overall efficacy of nifedipine was attributable to a high incidence of side effects, including an unusual symptom complex resembling erythromelalgia. These side effects led 45% (9/20) of the nifedipine patients to withdraw from the study within two weeks. By contrast, no patient (0/18) withdrew from the study within the first 2 weeks of propranolol therapy. We conclude that nifedipine is not an agent of first choice for the prophylaxis of migraine.

Processing of 125I-insulin by polarized cultured kidney cells.

Renal clearance of insulin is achieved by glomerular filtration and by passage from the postglomerular peritubular circulation into the renal interstitium. In the proximal tubule, filtered insulin binds to the apical membrane and is internalized and degraded while insulin in the interstitium is taken up by receptor-mediated endocytosis and degraded. To study these processes we have utilized cultured opossum kidney cells. These cells have proximal-like features and process insulin in a manner consistent with that described in vivo. To study apical and basolateral uptake and metabolism of insulin independently, cells were grown on filters suspended in culture wells. insulin was degraded to large insulin-size intermediates and low-molecular-weight products. This occurred whether the protein was internalized from the apical or basolateral pole of the cells. Analysis of the intermediate products by reverse-phase high-performance liquid chromatography revealed that products formed after apical or basolateral internalization were similar. Since products were preferentially released from the side of uptake, it is likely that apically and basolaterally internalized insulin is degraded in comparable organelles located in different regions of the cell. Most of the internalized insulin traversed the degradative pathway but some insulin followed a retroendocytic or minor transcytotic pathway. Degradation was inhibited by chloroquine, which also selectively increased the release of internalized insulin from the apical pole irrespective of the side of uptake. Thus while the polar degradative processes appear to be similar in nature, the polar exocytotic processes appear to be different.

Down-regulation of monocyte tissue factor mediated by tissue factor pathway inhibitor and the low density lipoprotein receptor-related protein.

Inflammatory mediators like bacterial lipopolysaccharide induce monocytes to express tissue factor (TF), the cell-surface protein that triggers the blood clotting cascade in hemostasis and thrombotic disease. The physiologic ligand for TF is the serine protease, factor VIIa (FVIIa), and the resulting bimolecular enzyme, TF/FVIIa, can be reversibly inhibited by tissue factor pathway inhibitor (TFPI). Culturing monocytic cells in the presence of both FVIIa and TFPI caused down-regulation of TF expression via reducing its half-life. To exert this effect, FVIIa had to be competent to bind both TF and TFPI, and TFPI had to contain the C-terminal domain required for binding to other cell-surface receptors, including the low density lipoprotein receptor-related protein (LRP). TF down-regulation by FVIIa plus TFPI was abrogated by the 39-kDa receptor-associated protein, which blocks binding of all known ligands to LRP. Furthermore, treatment with FVIIa plus TFPI caused monocyte TF to colocalize with alpha-adaptin, a component of clathrin-coated pits. Thus, in addition to reversibly inhibiting TF/FVIIa catalytic activity, TFPI also mediates the permanent down-regulation of cell-surface TF in monocytic cells via LRP-dependent internalization and degradation. This represents an unusual mechanism for receptor internalization, requiring ligand-dependent bridging of one cell-surface receptor (TF) to a second cell-surface receptor (LRP), the latter being capable of clathrin-mediated internalization.

(R)-(-)-[77Br]4-bromo-2,5-dimethoxyamphetamine labels a novel 5-hydroxytryptamine binding site in brain membranes.

(R)-(-)-[77Br]4-Bromo-2,5-dimethoxyamphetamine [(R)-(-)-[77Br] DOB] was synthesized to a high specific activity (1875 +/- 50 Ci/mmol) and used to label membrane-associated recognition sites in rat brain. (R)-(-)-[77Br]DOB displayed high affinity (KD = 0.60 +/- 0.08 nM) for a relatively low density of binding sites (Bmax = 1.2 +/- 0.08 pmol/g of tissue) in rat cortical membranes as compared with [3H]ketanserin (KD = 0.65 +/- 0.1 nM; Bmax = 6.2 +/- 0.6 pmol/g of tissue). Guanine, but not adenine, nucleotides were found to inhibit specific (R)-(-)-[77Br]DOB binding. GTP (10(-4) M) did not eliminate specific (R)-(-)-[77Br]DOB binding but caused a competitive inhibition of the radioligand. Drug competition studies of 5-hydroxytryptamine (5-HT) and related agents indicate that both putative agonists and antagonists display nanomolar potency for these sites. A significant correlation (p less than 0.01) exists between drug potencies for (R)-(-)-[77Br]DOB-labeled sites and both 5-HT2 (r = 0.64) and 5-HT1C (r = 0.68) binding sites. However, the sites do not appear to be identical. Moreover, a significant correlation exists between drug potencies for (R)-(-)-[77Br]DOB-labeled sites and human hallucinogenic drug potencies (r = 0.89; p less than 0.01). We conclude that (R)-(-)-[77Br]DOB labels a unique 5-HT recognition site in rat brain that does not coincide with previously described 5-HT binding site subtypes. The (R)-(-)-[77Br]DOB site does not appear to be a high affinity "state" of the 5-HT2 receptor but may label a subset of heterogeneous 5-HT2 recognition sites.