The involvement of urothelial alpha1A adrenergic receptor in controlling the micturition reflex.

The current study was undertaken in an attempt to characterize the functional properties of urothelial alpha1A adrenergic receptors, especially in modulating the micturition reflex. The expression of alpha1A receptors in rat bladder was analyzed by immunohistochemistry and Western blotting. As a functional study, we obtained continuous infusion cystometrograms in conscious rats using noradrenaline (NA) and subtype selective alpha1 adrenergic receptor antagonists, tamsulosin (alpha1A/alpha1D selective) and silodosin (alpha1A superselective). Alpha1A receptors were immunohistochemically detected in rat urothelium. Intravesical infusion of NA (60 microM) significantly shortened the intercontraction interval (ICI). Pretreatment with tamsulosin at a dose of 0.4 microg/kg i.v. abolished intravesical NA infusioninduced reduction of ICI. Neither intravesical infusion of tamsulosin (20 microM) nor that of silodosin (0.2 microM) significantly altered ICI. After intravesical infusion of silodosin, intravesical NA infusion did not affect ICI. Urothelial alpha1A receptors might modulate bladder afferent activity under pathophysiological conditions with augmented concentrations of NA in blood or urine.

Cellular expression of a sodium-dependent monocarboxylate transporter (Slc5a8) and the MCT family in the mouse kidney.

Expression analysis of transporters selective for monocarboxylates such as lactate and ketone bodies in the kidney contributes to understanding the renal energy metabolism. Distribution and expression intensity of a sodium-dependent monocarboxylate transporter (SMCT) and proton-coupled monocarboxylate transporters (MCT) were examined in the mouse kidney. In situ hybridization survey detected significant mRNA expressions of SMCT and MCT-1, 2, 5, 8, 9, 10, and 12. Among these, signals for SMCT, MCT2 and MCT8 were predominant; transcripts of SMCT were restricted to the cortex and the outer stripe of outer medulla, while those of MCT2 and MCT8 gathered in the inner stripe of outer medulla and the cortex, respectively. Immunohistochemically, SMCT was present at the brush border in S2 and S3 of proximal tubules, suggesting the active uptake of luminal monocarboxylates here. MCT1 and MCT2 immunoreactivities were respectively found baso-laterally in S1 and thick ascending limbs of Henle's loop. The cellular localization of transporters suggests the involvement of SMCT in the uptake of filtrated lactate and ketone bodies and that of MCTs in the transport of monocarboxylate metabolites between tubular cells and circulation, but the different distribution patterns do not support the notion of a functional linkage between SMCT and MCT1/MCT2.

Urothelium EP1 receptor facilitates the micturition reflex in mice.

We investigated the presence of EP1 receptor in the urothelium and its role in micturition reflex by examining the effect of intravesical administration of prostaglandin E(2) (PGE2), an EP1 agonist (ONO-DI-004), acetic acid, and capsaicin. Age-matched EP1-KO mice and C57BL/6 wild-type (WT) mice were used. Western blots and standard immunohistochemical procedures were performed. Cystometrygram (CMG) was performed without anesthesia in a restraining cage. ATP release from the cultured urothelium cells was performed using luciferin-luciferase luminometry. The EP1 receptor was found to be present in the urothelium. In WT mice, PGE2 infusion shortened the intercontraction interval (ICI) in a dose-dependent fashion; however, it did not alter the ICI in EP1-KO mice. The EP1 agonist significantly shortened the ICI in WT mice, but not in EP1-KO mice. Acetic acid and capsaicin shortened the ICI in both WT mice and EP1-KO mice. EP1 agonist, PGE2 and capsaicin provoked ATP release from cultured urothelial cells. These results suggest that EP1 receptor was present in bladder urothelium, and could be activated by PGE2 to release ATP. EP1 receptor in urothelium might be important for reflex voiding in pathological conditions.

Activation of alpha1D adrenergic receptors in the rat urothelium facilitates the micturition reflex.

PURPOSE: Previous studies have revealed that the activation of alpha(1) adrenergic receptors in urothelial cells releases neurotransmitters. We determined if alpha(1D) adrenergic receptors are expressed in the urothelium of the rat bladder and if inhibition of these receptors affects reflex voiding. MATERIALS AND METHODS: Female Wistar rats were used in the experiments. Receptor expression was evaluated by Western blot. The effects of receptor activation were studied using cystometrograms, measurement of adenosine triphosphate concentrations in the bladder lumen and afferent nerve recording. The alpha(1D) antagonist naftopidil (0.75 to 1.66 mg/kg) was administered intravenously into the external jugular vein. RESULTS: The expression of alpha(1D) adrenergic receptors was detected in urothelial tissue with Western blot and immunohistochemistry. The alpha(1D) receptor antagonist naftopidil prolonged the intercontraction interval during continuous infusion cystometrograms in conscious rats (143% of the control value) and suppressed the excitatory effect of intravesical infusion of acetic acid (0.1%) on the intercontraction interval (220%). Naftopidil inhibited the bladder afferent nerve activity induced by bladder distention (32.0%) and acetic acid infusion (30.4%), and decreased adenosine triphosphate levels in the bladder perfusate during bladder distention (36.6%). CONCLUSIONS: Endogenous catecholamines appear to act on alpha(1D) receptors in the urothelium to facilitate mechanosensitive bladder afferent nerve activity and reflex voiding.

Synaptic specializations exist between enteric motor nerves and interstitial cells of Cajal in the murine stomach.

Autonomic neurotransmission is thought to occur via a loose association between nerve varicosities and smooth muscle cells. In the gastrointestinal tract ultrastructural studies have demonstrated close apposition between enteric nerves and intramuscular interstitial cells of Cajal (ICC-IM) in the stomach and colon and ICC in the deep muscular plexus (ICC-DMP) of the small intestine. In the absence of ICC-IM, postjunctional neural responses are compromised. Although membrane specializations between nerves and ICC-IM have been reported, the molecular identity of these specializations has not been studied. Here we have characterized the expression and distribution of synapse-associated proteins between nerve terminals and ICC-IM in the murine stomach. Transcripts for the presynaptic proteins synaptotagmin, syntaxin, and SNAP-25 were detected. Synaptotagmin and SNAP-25-immunopositive nerve varicosities were concentrated in varicose regions of motor nerves and were closely apposed to ICC-IM but not smooth muscle. W/W(V) mice were used to examine the expression and distribution of synaptic proteins in the absence of ICC-IM. Transcripts encoding synaptotagmin, syntaxin, and SNAP-25 were detected in W/W(V) tissues. In the absence of ICC-IM, synaptotagmin and SNAP-25 were localized to nerve varicosities. Reverse transcriptase polymer chain reaction (RT-PCR) and immunohistochemistry demonstrated the expression of postsynaptic density proteins PSD-93 and PSD-95 in the stomach and expression levels of PSD-93 and PSD-95 were reduced in W/W(V) mutants. These data support the existence of synaptic specializations between enteric nerves and ICC-IM in gastric tissues. In the absence of ICC-IM, components of the synaptic vesicle docking and fusion machinery is trafficked and concentrated in enteric nerve terminals.

Intestinal surgical resection disrupts electrical rhythmicity, neural responses, and interstitial cell networks.

BACKGROUND & AIMS: Surgical manipulations of the gastrointestinal (GI) tract, including intestinal resection and anastomosis, lead to motility disorders including a decrease in phasic and segmental contractions. The aims of the present investigation were to determine mechanisms underlying the loss of motility in a murine model of intestinal resection and to follow-up the recovery of intestinal motility after surgical manipulation. METHODS: Segments of ileum were removed from mice and the intestines were reconstructed. After surgery, the structure and activity of the ileal muscles, 0-5 cm oral and aboral to the site of resection, were examined at 5 and 24 hours with intracellular microelectrode recordings, isometric force measurements, Kit-like immunohistochemistry, and electron microscopy. RESULTS: Five hours after surgery there was loss of electrical slow waves and phasic contractions in muscles near the site of resection. This defect decreased as a function of distance above and below the resection. Tissues in the affected region were poorly responsive to carbachol and transmural nerve stimulation. Kit-like immunohistochemistry revealed disruption in interstitial cell of Cajal (ICC) networks at the level of the myenteric and deep muscular plexuses. Lesions in ICCs decreased with distance from the site of resection. Slow waves and mechanical activity recovered at the site of anastomosis 24 hours after surgery and recovered more rapidly when tissues were incubated in the inducible nitric oxide synthase (iNOS) inhibitor, L-N6 -(1-Iminoethyl) lysine hydrochloride (L-NIL). CONCLUSIONS: Loss of intestinal motility after surgery is associated with acute disruption of ICC networks, slow waves, and phasic contractions. This activity partially recovered within 24 hours after surgery.

Production and characterization of monoclonal antibodies against midgut of ixodid tick, Haemaphysalis longicornis.

There are concerted efforts toward development of tick vaccines to replace current chemical control strategies that have serious limitations [Parasitologia 32 (1990) 145; Infectious Disease Clinics of North America (1999) 209-226]. In this study, monoclonal antibodies (mAbs) specific to Haemaphysalis longicornis midgut proteins were produced and characterized. Eight antibody-secreting hybridomas were cloned and the mAbs typed as IgG1, IgG2a and IgG2b. On immunoblots, all mAbs reacted with a midgut protein band of about 76 kDa. All mAbs uniformly immunogold-stained the surface or epithelial layers of H. longicornis midgut and endosomes. Adult ticks (50%) that fed on an ascitic mouse producing the IgGs developed a red coloration and did not oviposit. As such, the 76 kDa protein that reacted with the mAbs could, therefore, be a potential candidate for tick vaccine development.

Passive immunization with monoclonal antibodies: effects on Haemaphysalis longicornis tick infestation of BALB/c mice.

Tick vaccine development plays an important role in current tick control strategies. Previously, we have produced three different isotypes of monoclonal antibodies (mAbs) which recognized a midgut protein of adult Haemaphysalis longicornis. These mAbs, typed as IgG1, 2a, and 2b, reacted with a 76 kDa surface protein of midgut cells. We speculated that the 76 kDa protein may be an unknown antigen for a tick vaccine and the three mAbs may work as probes to clone the protein. In this study, to test whether these three isotypes have anti-tick effects and if so which works more effectively, we conducted passive immunization in BALB/c mice with each of the mAbs, and infested the mice with adult ticks. All isotypes significantly reduced the number of hatched larvae, compared to controls, however, no differences in the magnitude of the reduction were observed among the three.

Glucocorticoid inhibits expression of V-1, a catecholamine biosynthesis regulatory protein, in cultured adrenal medullary cells.

V-1 acts as a positive and coordinate regulator of gene expression of catecholamine biosynthetic enzymes in PC12D cells. The present study was conducted to investigate the mechanism controlling expression of V-1 in the adrenal gland. Immunohistochemical analysis demonstrated that noradrenergic chromaffin cells more highly expressed V-1 than adrenergic chromaffin cells preferentially expressing the glucocorticoid receptor in rat adrenal glands. Western blotting showed that in cultured bovine adrenal medullary cells, dexamethasone, a synthetic glucocorticoid, inhibited expression of V-1, and that this inhibition was prevented by RU-486, a glucocorticoid receptor antagonist. These results suggest that adrenal expression of V-1 is differentially controlled by glucocorticoids through the specific receptor, and that thereby V-1 regulates catecholamine biosynthesis in a catecholaminergic phenotype-dependent manner.

Epididymis is a novel site of erythropoietin production in mouse reproductive organs.

The epididymis consists of the interstitial tissue and the ductus epididymidis, an extremely tortuous duct, in which spermatozoa exported from the testis gain motility and fertilizing capacity. We found that the cultured mouse epididymis produces erythropoietin (Epo). The content of Epo mRNA in the epididymis from the adult mouse (8-week-old) amounts to 40% of that in the kidney. The epididymal Epo mRNA dramatically increased upon growth; its level increased 120-fold from the age of 3 weeks to 7 weeks when they complete sexual maturation, while the increase in the total RNA was 3-fold. Hypoxia induced a 5-fold increase in the epididymal Epo mRNA transiently, which is much lower than the induction in the kidney (28-fold). In situ hybridization technique elucidated that the site of Epo production was located in the interstitial space between ductus epididymidis. The epididymal Epo may have an unidentified function in the male reproductive organ.

Cellular localization of the diazepam binding inhibitor in glial cells with special reference to its coexistence with brain-type fatty acid binding protein.

The diazepam binding inhibitor (DBI) was originally isolated from the brain as an intrinsic ligand of the benzodiazepine binding site on the type-A gamma-aminobutyric acid receptor (GABA(A) receptor). Its wide-spread distribution in non-neural tissues outside the brain suggests that DBI has various functions other than GABA-mediated neurotransmission. Since DBI is identical with the acyl-CoA binding protein, which has the ability to bind long chain acyl-CoA esters, the major function of DBI may possibly be related to lipid metabolism. This idea was supported by our previous study showing the consistent coexpression of DBI and fatty acid binding proteins (FABPs) in epithelia throughout the gastrointestinal tract. The present histochemical study focused on the distribution of DBI in neural tissues, and revealed a definite existence of DBI in non-neuronal supporting cells in both the central and peripheral nervous systems. In the brain, intense immunoreactivity for DBI was detected in the cerebellar Bergmann glia, olfactory ensheathing glia, subgranular layer of the dentate gyrus, and retinal Muller cells. In the peripheral nervous system, satellite cells in sensory/autonomic ganglia, Schwann cells, and sustentacular cells in the adrenal medulla were immunoreactive to a DBI antibody. Moreover, the colocalization of DPI and brain-type FABP (B-FABP) was observed in most of the non-neuronal supporting cells mentioned above, indicating that DBI and B-FABP are cooperatively involved in the energy metabolism of astrocytes and related cells, which are thought to support neuronal development and functions.