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We have discovered high-affinity antagonists (exemplified by 11 and 12) that are the most selective for α1d-adrenergic receptors (α1d-AR) reported to date. In cloned receptor assay systems, 12 displays at least 95-fold selectivity for the α1d-AR over all other G-protein-coupled receptors tested, and the subtype selectivity of 11 was confirmed in pharmacologically defined isolated tissue preparations.

The role of N-linked oligosaccharide side chains in the biogenesis and function of Na+-coupled transporters in renal luminal brush-border membrane (BBM) is not known. We examined the question of how in vivo inhibition by alkaloid swainsonine of alpha-mannosidase, a key enzyme in processing of glycoproteins in the Golgi apparatus, affects Na+/H+ antiport and Na+/Pi symport as well as activities of other transporters and enzymes in rat renal BBM. Administration of swainsonine to thyroparathyroidectomized rats, control or treated with 3,5,3′-triiodothyronine, markedly decreased the rate of Na+/H+ antiport, but had no effect on the rate of Na+/Pi symport across renal BBM vesicles (BBMV). Moreover, administration of swainsonine did not change activities of Na+ gradient, ([extravesicular Na+] greater than [intravesicular Na+])-dependent transport of D-glucose, L-proline, or the amiloride-insensitive 22Na+ uptake by BBMV; the activities of the BBM enzymes alkaline phosphatase, gamma-glutamyltransferase, or leucine aminopeptidase in BBMV were also not changed. The in vitro enzymatic deglycosylation of BBM by incubating freshly isolated BBMV with bacterial endoglycosidase F also resulted in a decreased rate of Na+/H+ antiport, but not Na+-coupled symports of Pi, L-proline, and D-glucose, or the activities of the BBM enzymes were not significantly affected. Similar incubation with endoglycosidase H was without effect on any of these parameters. Both the modification of BBMV glycoproteins by administration fo swainsonine in vivo as well as the in vitro incubation of BBMV with endoglycosidase F resulted in a decrease of the apparent Vmax of Na+/H+ antiport, but did not change the apparent Km of this antiporter for extravesicular Na+ and did not increase H+ conductance of BBM. Taken together, our findings suggest that intact N-linked oligosaccharide chains of the biantennary complex type in renal BBM glycoproteins are required, directly or indirectly, for the transport function of the Na+/H+ antiporter inserted into BBM of renal proximal tubules.

We investigated interactions of phosphonoformic acid (PFA), phosphonoacetic acid (PAA), and other phosphonyl derivatives with the Na+ gradient [Na+ extravesicular greater than Na+ intravesicular; Nao+ greater than Na+i]-dependent transport system for phosphate (Pi) in renal cortical brush border membrane vesicles (BBMV). PFA and PAA inhibited in a dose-dependent manner the Na+ gradient [Na+o greater than Na+i]-dependent uptake of Pi by rat kidney BBMV. PFA was a more potent inhibitor than PAA while phosphonopropionic acid, hydroxymethylphosphonic acid, and phenylphosphonic acid had no effect on Pi transport. The inhibitory effect of PFA was competitive (Ki approximately equal to 4.6 X 10(-4) M) and reversible upon dilution. The uptake of Pi by BBMV in the absence of Na+ gradient [Nao+ = Na+i] was also inhibited by PFA. The PFA had no effect on uptake of L-[3H]proline, D-[3H]glucose, or 22Na+ by BBMV nor did it alter intravesicular volume of BBMV. The relative (%) extent of inhibition by PFA was not altered by changes in the extravesicular pH or changes in the steepness of the Na+ gradient [Nao+ greater than Na+i]. The inhibition of PFA was analogous in renal BBMV from rats, mice, rabbits, or dogs. Unlike other known inhibitors of brush border membrane (BBM) transport of Pi, e.g. arsenate, NAD, and ethane-1-hydroxy-1,1-diphosphonate, PFA and PAA had no inhibitory effect on BBM-bound or solubilized alkaline phosphatase. Also, PFA did not interfere with the activity of renal cortical (Na-K)ATPase. Administration of PFA (0.5 g/kg/day, intraperitoneally) to thyroparathyroidectomized rats fed a low Pi diet elicited an increase in urinary excretion of Pi, but did not change the excretion of Na+, K, and Ca2+. The results show that the PFA, and to a lesser degree PAA, are specific competitive inhibitors of the Na+-Pi cotransport in renal cortical BBM and are suitable probes for studies of this transport system.

Campylobacter jejuni, a microaerophilic, gram-negative bacterium, is a common cause of gastrointestinal disease in humans. Heat shock proteins are a group of highly conserved, coregulated proteins that play important roles in enabling organisms to cope with physiological stresses. The primary aim of this study was to characterize the heat shock response of C. jejuni. Twenty-four proteins were preferentially synthesized by C. jejuni immediately following heat shock. Upon immunoscreening of Escherichia coli transformants harboring a Campylobacter genomic DNA library, one recombinant plasmid that encoded a heat shock protein was isolated. The recombinant plasmid, designated pMEK20, contained an open reading frame of 1,119 bp that was capable of encoding a protein of 372 amino acids with a calculated molecular mass of 41,436 Da. The deduced amino acid sequence of the open reading frame shared similarity with that of DnaJ, which belongs to the Hsp-40 family of molecular chaperones, from a number of bacteria. An E. coli dnaJ mutant was successfully complemented with the pMEK20 recombinant plasmid, as judged by the ability of bacteriophage lambda to form plaques, indicating that the C. jejuni gene encoding the 41-kDa protein is a functional homolog of the dnaJ gene from E. coli. The ability of each of two C. jejuni dnaJ mutants to form colonies at 46 degreesC was severely retarded, indicating that DnaJ plays an important role in C. jejuni thermotolerance. Experiments revealed that a C. jejuni DnaJ mutant was unable to colonize newly hatched Leghorn chickens, suggesting that heat shock proteins play a role in vivo.

Twenty-one isolates were tested for their ability to adhere to and invade HEp-2 cells in vitro. Of the 21 organisms tested, 2 did not invade the HEp-2 cells, and 1 of these did not adhere to the epithelial cells. Campylobacter jejuni clinical isolates were more invasive than the nonclinical strains that were tested. When HEp-2 cells were treated with cytochalasin B, the invasiveness of C. jejuni was reduced, indicating active participation of the host cell in the uptake of these organisms. The number of intracellular C. jejuni isolates decreased when Campylobacter whole-cell lysates were absorbed onto HEp-2 cell monolayers. Experiments were also conducted to identify the functional sites of the antigens responsible for expression of Campylobacter invasion. Oxidation of lysates with sodium meta-periodate significantly affected its inhibitory capacity. This implies that the Campylobacter invasive ligand appears to be dependent upon an intact carbohydrate moiety.

Since phosphonoformic acid (PFA) acts as a specific competitive inhibitor of Na+-Pi co-transport across renal brush-border membrane (BBM), we employed the [14C]PFA as a probe to determine the mechanism of its interaction with rat renal BBM. The binding of [14C]PFA to BBM vesicles (BBMV), with Na+ present in extravesicular medium (Na+o), was time- and temperature-dependent. The replacement of Na+o with other monovalent cations reduced the PFA binding by −80%. Cl- was the most effective accompanying monovalent anion as NaCl for maximum PFA binding. The Na+o increased the apparent affinity of BBMV for [14C]PFA binding, but it did not change the maximum binding capacity. The maximum [14C]PFA binding was achieved at Na+o approximately equal to 50 mM. The extent of Na+-dependent [14C]PFA binding correlated (r = 0.98; p less than 0.01) with percent inhibition by an equimolar dose of PFA of the (Na+o greater than Na+i)-dependent BBMV uptake of 32Pi. Intravesicular Na+ (Na+i) decreased [14C]PFA binding, on BBMV, and this inhibition by Na+i was dependent on the presence of Na+o. The increase in Na+i, at constant [Na+]o, decreased the Vmax, but not the Km, for [14C]PFA binding on BBMV. Bound [14C]PFA was displaced from BBMV by phosphonocarboxylic acids proportionally (r = 0.99; p less than 0.05) to their ability to inhibit (Na+o greater than Na+i)-gradient-dependent Pi transport, whereas other monophosphonates, diphosphonates, L-proline, or D-glucose did not influence the [14C]PFA binding. The Na+-dependent binding of [14C]PFA and of [3H]phlorizin by BBMV was 10 times higher than binding of these ligands to renal basolateral membranes and to mitochondria. [14C]PFA probably binds onto the same locus on the luminal surface of BBM, where Pi and Na+ form a ternary complex with the Na+-Pi co-transporter.

Gizzard myosin, fluorescently labeled with tetramethylrhodamine iodoacetamide, was microinjected into living 3T3 fibroblasts to label myosin-containing structures. The fluorophore was located predominantly on the heavy chain near the COOH terminus of the S1 head and on the 17-kD light chain. After microinjection of a tracer amount into living 3T3 cells, the fluorescent myosin showed a distribution identical to that revealed by immunofluorescence with antimyosin antibodies. Injected myosin became localized in small beads, which were found along large stress fibers, along fine fibers, and in a poorly organized form near the lamellipodia. De novo assembly of beads was observed continuously within or near the lamellipodia, suggesting that myosin molecules may undergo a constant cycling between polymerized and unpolymerized states. The nascent structures then moved away from lamellipodia and became organized into linear arrays. Similar movement was also observed for beads already associated with linear structures, and may represent a continuous flux of myosin structures. The dynamic reorganization of myosin may play an important role in cell movement and polarity.