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PubMed2000

Intact endothelial and smooth muscle function in small resistance arteries after 48 h in vessel culture.

Long-term culture of resistance vessels allows introduction of molecular biology techniques for use in microvascular research. The aim of the present study was to establish a culture protocol that preserved vascular integrity and function in microvessels for 48 h in culture. Skeletal muscle resistance arteries were excised from the hamster gracilis muscle. Segments were assigned to immediate functional tests or to vessel culture, during which segments were perfused and superfused at a transmural pressure of 45 mmHg with Leibovitz (L15) medium containing 15% fetal calf serum and antibiotics for 48 h. Cultured and freshly isolated vessels showed similar levels of spontaneous tone, myogenic responses, changes in smooth muscle intracellular calcium (Ca(i)(2+)) (fura 2), and vascular diameter (video microscopy) in response to 0.3 M norepinephrine and similar concentration-response curves for acetylcholine (endothelium dependent, +/-N(omega)-nitro-L-arginine) and sodium nitroprusside (endothelium independent). Measurements of endothelial Ca(i)(2+) revealed similar acetylcholine-induced increases in endothelial Ca(i)(2+) in both groups. It is concluded that vascular function can be preserved while maintaining vessels in culture. Thus it is possible to utilize protocols that require long-term treatment.

Animals↗
PubMed2000

Vasodilator mechanisms in the coronary circulation of endothelial nitric oxide synthase-deficient mice.

Previous studies have demonstrated that responses to endothelium-dependent vasodilators are absent in the aortas from mice deficient in expression of endothelial nitric oxide synthase (eNOS -/- mice), whereas responses in the cerebral microcirculation are preserved. We tested the hypothesis that in the absence of eNOS, other vasodilator pathways compensate to preserve endothelium-dependent relaxation in the coronary circulation. Diameters of isolated, pressurized coronary arteries from eNOS -/-, eNOS heterozygous (+/-), and wild-type mice (eNOS +/+ and C57BL/6J) were measured by video microscopy. ACh (an endothelium-dependent agonist) produced vasodilation in wild-type mice. This response was normal in eNOS +/- mice and was largely preserved in eNOS -/- mice. Responses to nitroprusside were also similar in arteries from eNOS +/+, eNOS +/-, and eNOS -/- mice. Dilation to ACh was inhibited by N(G)-nitro-L-arginine, an inhibitor of NOS in control and eNOS -/- mice. In contrast, trifluoromethylphenylimidazole, an inhibitor of neuronal NOS (nNOS), decreased ACh-induced dilation in arteries from eNOS-deficient mice but had no effect on responses in wild-type mice. Indomethacin, an inhibitor of cyclooxygenase, decreased vasodilation to ACh in eNOS-deficient, but not wild-type, mice. Thus, in the absence of eNOS, dilation of coronary arteries to ACh is preserved by other vasodilator mechanisms.

Acetylcholine↗
PubMed2000

Myofilament lattice spacing as a function of sarcomere length in isolated rat myocardium.

The Frank-Starling relationship of the heart has, as its molecular basis, an increase in the activation of myofibrils by calcium as the sarcomere length increases. It has been suggested that this phenomenon may be due to myofilaments moving closer together at longer lengths, thereby enhancing the probability of favorable acto-myosin interaction, resulting in increased calcium sensitivity. Accordingly, we have developed an apparatus so as to obtain accurate measurements of myocardial interfilament spacing (by synchrotron X-ray diffraction) as a function of sarcomere length (by video microscopy) over the working range of the heart, using skinned as well as intact rat trabeculas as model systems. In both these systems, lattice spacing decreased significantly as sarcomere length was increased. Furthermore, lattice spacing in the intact muscle was significantly smaller than that in the skinned muscle at all sarcomere lengths studied. These observations are consistent with the hypothesis that lattice spacing underlies length-dependent activation in the myocardium.

Actin Cytoskeleton↗
PubMed2000

Human coronary arteriolar dilation to adrenomedullin: role of nitric oxide and K(+) channels.

Adrenomedullin (ADM) is a vasodilator produced by vascular endothelium and smooth muscle cells. Although plasma ADM levels are increased in patients with hypertension, heart failure, and myocardial infarction, little information exists regarding the microvascular response to ADM in the human heart. In the present study we tested the hypothesis that ADM produces coronary arteriolar dilation in humans and examined the mechanism of this dilation. Human coronary arterioles were dissected and cannulated with micropipettes. Internal diameter was measured by video microscopy. In vessels constricted with ACh, the diameter response to cumulative doses of ADM (10(-12)-10(-7) M) was measured in the presence and absence of human ADM-(22-52), calcitonin gene-related peptide-(8-37), N(omega)-nitro-L-arginine methyl ester (L-NAME), indomethacin (Indo), (1)H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one, SQ-22536, or KCl (60 mM). ADM dilated human coronary arterioles through specific ADM receptors (maximum dilation = 69 +/- 11%). L-NAME or N-monomethyl-L-arginine attenuated dilation to ADM (for L-NAME, maximum dilation = 66 +/- 7 vs. 41 +/- 13%, P < 0.05). Thus the mechanism of ADM-induced dilation involves generation of nitric oxide. However, neither (1)H-[1,2,4]oxadiazolo-[4, 3-a]quinoxalin-1-one, SQ-22536, nor Indo alone altered dilation to ADM. High concentrations of KCl blocked dilation to ADM. The magnitude of ADM dilation was reduced in subjects with hypertension. We propose that, in human coronary arterioles, ADM elicits vasodilation in part through production of nitric oxide and in part through activation of K(+) channels, with little contribution from adenylyl cyclase. The former dilator mechanism is independent of the more traditional pathway involving activation of soluble guanylate cyclase.

Adenine↗
PubMed2001

Focal topographic changes in inflammatory microcirculation associated with lymphocyte slowing and transmigration.

Microcirculation is the primary mechanism for delivering lymphocytes to inflammatory tissues. Blood flow within microvessels ensures a supply of lymphocytes at the blood-endothelial interface. Whether the structure of the inflammatory microcirculation facilitates lymphocyte transmigration is less clear. To illuminate the microcirculatory changes associated with lymphocyte transmigration, we used intravital videomicroscopy to examine the dermal microcirculation after application of the epicutaneous antigen oxazolone. Intravascular injection of fluorescein-labeled dextran demonstrated focal topographic changes in the microcirculation. These focal changes had the appearance of loops or hairpin turns in the oxazolone-stimulated skin. Changes were maximal at 96 h and coincided with peak lymphocyte recruitment. To determine whether these changes were associated with lymphocyte transmigration, lymphocytes obtained from efferent lymph of draining lymph nodes at 96 h were fluorescently labeled and reinjected into inflammatory microcirculation. Epifuorescence intravital video microscopy demonstrated focal areas were associated with lymphocyte slowing and occasional transmigration. In contrast, focal loops and lymphocyte slowing were rarely observed in the contralateral control microcirculation. Results suggest that structural adaptations in inflammatory microcirculation represented by focal topographic changes may contribute to regulation of tissue entry by recirculating lymphocytes.

Adjuvants, Immunologic↗
PubMed1992

Bioassay of a tracheal smooth muscle-constricting factor released by respiratory epithelial cells.

Epithelium-derived factors of unknown identity have been proposed to modulate airway smooth muscle tone. We developed a novel sensitive bioassay system that allows serial perfusion of cultured respiratory epithelial cells and guinea pig trachea (GPT). GPT responses were assessed as diameter changes by computerized video microscopy (resolution, 15 microns). A permanent hamster lung epithelial cell line was grown on microcarrier beads and perfused in a cell column. When the outflow tubing from the epithelial cell column was connected to the inflow cannula, the detector GPT contracted, reaching 28 +/- 6% of the maximum methacholine (100 microM)-induced contraction (n = 12, P less than 0.001). Perfusion of the cell column with diclofenac (10 microM) or lysin-mono-acetylsalicylic acid (100 microM) abolished the GPT contraction, whereas selective perfusion of the detector GPT with either agent did not block the contraction. Analysis of the effluent of the epithelial cell column demonstrated a significant basal release of prostaglandins F2 alpha and E2 (PGF2 alpha and PGE2) and 6-ketoprostaglandin F1 alpha, whereas only marginal amounts of thromboxane B2 were detected. When given exogenously, PGF2 alpha, PGE2, PGI2, and U-46619 all contracted the GPT. It is concluded that lung epithelial cells can contract GPT by releasing a transferable factor. This factor is likely to be a constrictor cyclooxygenase product that is not produced in epithelium-denuded GPT.

Animals↗
PubMed1994

Transforming growth factor-alpha enhances alveolar epithelial cell repair in a new in vitro model.

Alveolar epithelial type II cells are essential for regenerating an intact alveolar barrier after destruction of type I cells in vivo. The first objective of these experimental studies was to develop an in vitro model to quantify alveolar epithelial cell wound repair. The second objective was to investigate mechanisms of alveolar epithelial cell wound healing by studying the effects of serum and transforming growth factor-alpha (TGF-alpha) on wound closure. Primary cultures of rat alveolar type II cells were prepared by standard methods and grown to form confluent monolayers in 48 h. Then a wound was made by denuding an area (mean initial area of 2.1 +/- 0.6 mm2) of the monolayer. Re-epithelialization of the denuded area over time in the presence or absence of serum was measured using quantitative measurements from time-lapse video microscopy. The half time of wound healing was significantly enhanced in the presence of serum compared with serum-free conditions (2.4 +/- 0.2 vs. 17.4 +/- 0.8 h, P < 0.001). We then tested the hypothesis that TGF-alpha is an important growth factor for stimulating wound repair of alveolar epithelial cells. Exogenous addition of TGF-alpha in serum-free medium resulted in a significantly more rapid wound closure, and, furthermore, the addition of a monoclonal antibody to TGF-alpha in the presence of serum significantly decreased fourfold the rate of wound closure. Measurement of internuclear cell distance confirmed that both cell motility and cell spreading were responsible for closure of the wound. These data demonstrate that 1) the mechanisms of alveolar cell repair can be studied in vitro and that 2) TGF-alpha is a potent growth factor that enhances in vitro alveolar epithelial cell wound closure.

Animals↗
PubMed1995

TNF-alpha and IL-1 beta upregulate nitric oxide-dependent ciliary motility in bovine airway epithelium.

Airway epithelial cells can be modulated by cytokines such as tumor necrosis factor (TNF)-alpha and interleukin (IL)-1 beta that are released from inflammatory cells. Since ciliary motility is an important host defense function of airway epithelium, we hypothesized that cytokines, released from lung macrophages, upregulate ciliary motility. To test this hypothesis, ciliary beat frequency (CBF) was measured by video microscopy in cultured ciliated bovine bronchial epithelial cells (BBECs) incubated for 24 h with bovine alveolar macrophage-conditioned medium (AM-CM). Exposure to AM-CM resulted in a delayed (> or = 2 h) increase in CBF that was maximal after 24 h exposure (13.70 +/- 0.43 for AM-CM vs. 9.44 +/- 0.24 Hz for medium; P < 0.0001) and which was largely blocked by either anti-TNF-alpha or anti-IL-1 beta antibodies. rTNF-alpha or rIL-1 beta similarly increased CBF, which could be blocked by preincubation with either anti-rTNF-alpha or anti-rIL-1 beta antibodies. Preincubation of BBECs with actinomycin D or dexamethasone also blocked rTNF-alpha- and rIL-1 beta-induced cilia stimulation, suggesting that new protein synthesis is required for cytokine-induced upregulation of CBF. Since NO is known to upregulate ciliary motility and cytokines can induce NO synthase (NOS), we hypothesized that TNF-alpha and IL-1 beta increase CBF by inducing NOS in BBECs. The cilia stimulatory effects of TNF-alpha or IL-1 beta were inhibited by NG-monomethyl-L-arginine, a competitive NOS inhibitor, and restored by the addition of either L-arginine, an NOS substrate, or sodium nitroprusside, an NO donor.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Oxidoreductases↗
PubMed1995

Regulation of fluid-phase endocytosis in alveolar macrophages.

We investigated whether fluid-phase endocytosis in rabbit alveolar macrophages (AM) was regulated by alterations in intracellular adenosine 3',5'-cyclic monophosphate (cAMP). Suspensions of freshly isolated AM were incubated with anionic dextrans (mol mass = 10 kDa), coupled to fluorescein isothiocyanate (FITC), at either 37 or 4 degrees C. There was a rapid increase in AM-associated fluorescence, quantified by laser flow-cytometry and video microscopy during the first hour of incubation at 37 degrees C, which was directly proportional to the amount of tracer present in the medium. In contrast, at 4 degrees C, AM fluorescence was similar to autofluorescence. Incubation of AM with forskolin (50 microM) or 3-isobutyl-1-methyl xanthine (IBMX; 0.1 mM) increased their cAMP content by 67 +/- 2 and 52 +/- 5% (mean +/- SE; n = 4) and decreased FITC-dextran uptake by 29 +/- 4 and 31 +/- 4% (n = 3). On the other hand, incubation of AM with 0.5 mM IBMX inhibited FITC-dextran uptake by 62 +/- 4% (n = 3), without any further increase in cAMP. Incubation of AM with 0.4 mM 8-(4-chlorophenylthio)-adenosine 3',5'-cyclic monophosphate (CPT-cAMP), a cell-permeable analogue of cAMP, decreased FITC-dextran uptake by 48 +/- 5% (n = 6). Pulse-chase experiments showed that the rate of FITC-dextran exocytosis was not affected by cAMP. We concluded that fluid-phase endocytosis in rabbit AM is regulated by cAMP and by an additional, cAMP-independent mechanism of IBMX.

1-Methyl-3-isobutylxanthine↗
PubMed1993

Microcirculatory effects of hypoxic and hypercapnic vasoconstriction in frog skin.

The effects in frog (albino Xenopus laevis) skin of hypoxic and hypercapnic vasoconstriction on the following microcirculatory parameters were determined: capillary red blood cell flux, capillary red blood cell velocity, perfused capillary density, lineal red blood cell density, and the temporal heterogeneity of capillary red blood cell velocities. All of these parameters affect the gas exchange characteristics of respiratory organs. Measurements were made by fluorescent video microscopy of a 1.5-cm2 region of skin exposed to different gas mixtures (air, O2, N2, 5% CO2-95% air, 5% CO2-95% N2). N2 caused red blood cell flux and velocity to fall to 52 +/- 10% (P < 0.05) and 47 +/- 10% (P < 0.01), respectively, of those values during air exposure. Five percent CO2 caused capillary red blood cell flux and velocity to decrease by 51 +/- 11% (P < 0.05) and 43 +/- 11% (P < 0.01), respectively. Fluxes (P < 0.01) and velocities (P < 0.01) were also less with 5% CO2-95% N2 than with air. There were no significant differences in flux and velocity between N2, 5% CO2-95% air and 5% CO2-95% N2 (P > 0.1). There was no significant difference in flux or in velocity between O2 and air (P > 0.1). Gas composition had no significant effect on lineal red blood cell density (P > 0.35) or the density of perfused capillaries (P > 0.22). The heterogeneity of cell velocities was significantly greater with N2 than with the other gases (P < 0.01). There was no significant difference in red blood cell velocity heterogeneity between the other gases (P > 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Nocodazole inhibition of organic anion secretion in teleost renal proximal tubules.

The impact of the microtubule-disrupting drug nocodazole on renal tubular secretion of organic anions was examined in vitro using proximal tubular masses from teleost fish. Nocodazole reversibly inhibited 20-30% of the tubular accumulation of two model organic anions, p-aminohippurate and fluorescein (FL), by winter flounder tubular masses. However, the drug had no effect on the initial rate of organic anion uptake. Thus it did not reduce transport into the cells at the basolateral membrane, either directly by affecting basolateral organic anion transport proteins or indirectly by altering metabolism or ion gradients. Instead, epifluorescence video microscopy and digital image analysis of killifish tubules showed that nocodazole greatly reduced luminal accumulation of FL and had a smaller effect on cellular dye accumulation. Luminal FL accumulation returned to control levels when tubules were incubated in drug-free medium. Confocal fluorescence microscopy confirmed the marked reduction in luminal FL concentration and demonstrated that intracellular punctate FL accumulation was also markedly reduced. Finally, immunohistochemistry with an anti-tubulin antibody showed that the concentrations of nocodazole used in the above experiments reversibly disrupted microtubules within renal epithelial cells. These data indicate that a component of organic anion secretion in teleost proximal tubule is dependent on an intact microtubular network.

Animals↗
PubMed1998

Regulation of the vasomotor activity of lymph microvessels by nitric oxide and prostaglandins.

It was shown previously that the presence of endothelium modulates spontaneous vasomotion of small lymphatic vessels. In the present study, we aimed to elucidate the nature of endothelium-derived factors, produced in basal conditions and in response to agonists, that affect the smooth muscle tone of lymph microvessels in vitro. Afferent lymph microvessels were isolated from rat iliac lymph nodes, cannulated with glass micropipettes, and pressurized (6 cmH2O), and changes in their diameter were investigated with video microscopy. In resting conditions, isolated lymph vessels exhibited spontaneous constrictions and dilations. The maximum and minimum diameters (Dmax and Dmin) were 149.8 +/- 2.9 and 85.8 +/- 3.6 microns, respectively. Acetylcholine (ACh, 10(-7) to 10(-5) M) and sodium nitroprusside (SNP, 10(-8) to 10(-6) M) temporarily abolished diameter oscillations, increasing the diameter of lymphatics dose dependently. For example, 10(-5) M ACh and 10(-6) M SNP increased the diameter (Dmax) by 15.2 +/- 2.2 and 25.0 +/- 2.7 microns, respectively. Treatment of vessels with NG-nitro-L-arginine (10(-4) M) significantly reduced the amplitude of diameter oscillations and nearly completely eliminated ACh-induced dilation of lymph microvessels, whereas SNP (10(-6) M) elicited a significantly greater dilation (55.6 +/- 7.5 microns). Arachidonic acid (AA, 10(-8) to 10(-6) M) constricted (up to 50 microns), whereas prostaglandin E2 (PGE2, 10(-9) to 10(-7) M) dilated (up to 40 microns), lymphatic vessels. Indomethacin (10(-5) M) increased both Dmax and Dmin and completely inhibited AA-induced constrictions, but did not affect PGE2-induced dilations of lymph microvessels. AA-induced constrictions of lymphatics were converted into dilations after treatment with SQ-29,548, a selective PGH2-thromboxane A2 (PGH2-TxA2, 10(-6) M) receptor antagonist, whereas PGE2-induced dilations were not affected. We conclude that endothelial nitric oxide and prostaglandins are important modulators of lymphatic vasomotion, hence pumping activity of lymph microvessels in vivo.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed2001

Sites and ionic mechanisms of hypoxic vasoconstriction in frog skin.

We tested the hypothesis that the cellular mechanisms mediating hypoxic vasoconstriction (HVC) in frog skin, an important vertebrate respiratory organ, are similar to those mediating HVC in the pulmonary vasculature of mammals. An accepted hypothesis in the lung is that alveolar hypoxia alters the redox potential in vascular smooth muscle cells of arterial vessels. This decreases membrane K+ conductance, causing depolarization. Depolarization increases the open probability of L-type Ca2+ channels, facilitating Ca2+ entry into the cell, which leads to vascular smooth muscle contraction and vasoconstriction. We studied the cutaneous microcirculation of the frog (Xenopus laevis) web by enclosing the web in a transparent chamber that was ventilated with different gas mixtures. Arteriolar and venular diameters were measured by video microscopy. Drugs were applied topically or intravascularly. A dose-dependent constriction to hypoxia occurred in arterioles but not venules, although both vessel types constricted to similar degrees to the thromboxane mimetic U-46619. The magnitude of HVC was not associated with arteriolar size. Constriction of arterioles with 4-amino pyridine, a K+-channel antagonist, was blocked by the L-type Ca2+-channel blocker nifedipine. Nifedipine also antagonized HVC and hypercapnic vasoconstriction. Bay K 8664, a drug that increases the open probability of L-type Ca2+ channels, augmented HVC. These data support our hypothesis that the cellular mechanisms mediating HVC are similar in frog skin and mammalian lungs. This similarity between amphibian and mammalian tissues suggests that the mechanisms of HVC may have arisen relatively early in vertebrate evolution. In addition, because of its structural simplicity and easy accessibility, frog skin may be a useful tissue for studying this general phenomenon in vivo.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Basolateral transport pathways for K+ and Cl- in rabbit proximal tubule: effects on cell volume.

To characterize the nature of K+ and Cl- transport pathways across basolateral membrane of rabbit proximal convoluted tubule, we used quantitative video microscopy to measure cell volume changes induced by rapid basolateral K+ and Cl- concentration changes. Elevating basolateral K+ resulted in cell swelling, which was largely inhibited by replacement of basolateral Cl- with cyclamate (85%) or by addition of 2 mM Ba2+ (72%). Substitution of basolateral Cl- by NO3- enhanced cell swelling, whereas substitution of Cl- by I- did not affect the K(+)-induced volume changes. Removal of Cl- from the bath reversed the cell swelling induced by raising K+ in the bath. Steady-state cell volume was 28% greater in hypotonic medium (250 mosmol/kgH2O) than in hypertonic medium (350 mosmol/kgH2O), and the rate of increase in cell volume induced by raising K+ was three times higher in hypotonic than in hypertonic medium. Substitution of Cl- by NO3- did not alter the effect of medium osmolality on K(+)-induced cell swelling, whereas addition of 0.2 mM diphenylamine-2-carboxylate inhibited the response (63%). We conclude that K(+)-induced cell swelling results from entry of K+ and Cl- into the cell across the basolateral membrane; it is proposed that transport of KCl across the basolateral cell membrane proceeds largely through two separate conductive pathways for K+ and Cl-. Cell swelling activates KCl transport occurring via K+ and Cl- channels across the basolateral membrane.

Animals↗
PubMed1992

Participation of protein kinase C in desensitization to bradykinin and to carbachol in MDCK cells.

To explore the possibility that protein kinase C (PKC) participates in desensitization to Ca(2+)-mobilizing hormones in MDCK cells, we measured intracellular free Ca2+ concentration ([Ca2+]i) using fura-2 and video microscopy. We first examined the response of MDCK cells grown on plastic dishes. Exposure of cells to bradykinin (BK) or to carbachol, followed by reexposure after washing off the hormone, revealed two features of hormone desensitization. First, the initial hormone-induced peak response of [Ca2+]i was transitory; [Ca2+]i returned to control levels despite continued presence of hormone. Second, cells remained refractory to hormone rechallenge for 5 min after washing off hormone; [Ca2+]i response on re-exposure was reduced 70% compared with initial hormone-stimulated peak. Subsequent experiments demonstrated involvement of PKC in both desensitization processes. Pretreatment with the phorbol ester, phorbol 12-myristate 13-acetate, significantly blunted initial response to BK and to carbachol by 70 and 86%, respectively. When hormone-stimulated C kinase activity was enhanced with the diglyceride lipase inhibitor, RG 80267, BK- and carbachol-induced increases in [Ca2+]i were blunted 50%. Pretreatment with sphingosine, an inhibitor of PKC, resulted in an amplification of initial hormone-stimulated increase in [Ca2+]i and restored the response to rechallenge. To examine the possible interaction between BK and carbachol,both of which use PKC to induce desensitization, we measured [Ca2+]i in cells grown as monolayers on permeable, collagen-coated supports. Both carbachol and BK induced desensitization to the other hormone (heterologous desensitization)provided that the two hormones were applied to the same side of the polarized monolayer (apical).(ABSTRACT TRUNCATED AT 250 WORDS)

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1992

Effects of ATP on pre- and postglomerular juxtamedullary microvasculature.

Based on evidence that extracellular ATP can influence vascular smooth muscle function in other organ systems, experiments were conducted to characterize the responsiveness of rat juxtamedullary microvascular segments to ATP. Experiments were performed using the in vitro blood-perfused juxtamedullary nephron preparation combined with video microscopy. Pentobarbital-anesthetized rats were pretreated with enalaprilat (2 mg iv) for 30 min before the right kidney was isolated and prepared for study. Renal perfusion pressure was set at 110 mmHg and held constant. Under control conditions, afferent and efferent arteriolar diameters averaged 19.9 +/- 1.4 (n = 19) and 21.6 +/- 1.2 microns (n = 10), respectively. Superfusion with 1, 10, and 100 microM ATP solutions induced sustained dose-dependent afferent vasoconstriction of 8.3 +/- 1.4, 12.8 +/- 1.7, and 12.1 +/- 2.1%, respectively (P < 0.01). Afferent vasoconstrictor responses to ATP were also observed during adenosine receptor blockade. In contrast, efferent arterioles were unresponsive to ATP stimulation even at concentrations as high as 100 microM (P > 0.05). Arcuate and interlobular arterial diameters averaged 82.0 +/- 15.7 (n = 5) and 43.4 +/- 4.5 microns (n = 6), respectively, during control conditions and responded to ATP treatment with a transient vasoconstriction followed by a gradual return to control diameter. Interlobular arteries exhibited a sustained constriction only at the 100 microM concentration (P < 0.05). These data demonstrate that afferent arterioles are more responsive to ATP treatment than other renal microvascular segments and suggest the presence of ATP-sensitive P2x purinoceptors on pre- but not postglomerular juxtamedullary microvascular elements.

Adenosine↗
PubMed1992

EDRF-angiotensin II interactions in rat juxtamedullary afferent and efferent arterioles.

The in vitro blood-perfused juxtamedullary nephron technique was utilized to determine the contribution of endothelium-derived relaxing factor (EDRF) to resting renal arteriolar caliber and to evaluate the interaction between EDRF and angiotensin II (ANG II) in renal microvascular control. Video microscopy was employed to visualize rat afferent and efferent arterioles and to measure their responses to blockade of nitric oxide (NO), which has been shown to account for much of the biological action of EDRF. The NO synthesis inhibitor, N omega-nitro-L-arginine (L-NNA), elicited vasoconstriction in a concentration-dependent manner, with 1,000 microM L-NNA significantly reducing both afferent (16 +/- 3%) and efferent (13 +/- 1%) diameters. This concentration of L-NNA also blocked the vasodilator response to 10 microM acetylcholine, while responsiveness to sodium nitroprusside was maintained. Vasoconstrictor responses to 1,000 microM L-NNA were attenuated in kidneys from rats pretreated with enalaprilat or losartan, reducing afferent diameter by 7 +/- 1 (n = 8) and 3 +/- 1% (n = 10) of control, respectively. Efferent arteriolar responses to L-NNA were similarly attenuated by losartan. The constrictor response to 10 nM ANG II was not exaggerated by L-NNA, suggesting that ANG II does not stimulate EDRF synthesis. These observations indicate that EDRF is continuously released in a quantity sufficient to affect both afferent and efferent arterioles of juxtamedullary nephrons in vitro. Furthermore, ANG II blockade attenuates the vasoconstriction elicited by L-NNA, suggesting that EDRF interacts with the renin-angiotensin system to control juxtamedullary afferent and efferent arteriolar resistance.

Acetylcholine↗
PubMed1994

Acidification of vasopressin-induced endosomes in toad urinary bladder.

It is well established that water channels (WC) are removed from the apical membrane of vasopressin-sensitive epithelia by endocytosis. The processing and the ultimate fate of endocytosed WC is, however, incompletely understood. In many cells, endosome acidification plays an important role in the processing and sorting of endocytosed proteins. Endosome acidification in the toad urinary bladder was therefore examined in vivo by fluorescence ratio video microscopy after induction of endocytosis by vasopressin removal and transepithelial water flow in the presence of the pH-sensitive fluid phase marker 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-dextran. Fifteen minutes after induction of endocytosis, the majority of endosomes had a neutral or slightly acidic pH. The number of acidic endosomes increased progressively with time. Two hours after endocytosis began, 98% of the endosomes had a pH < 6.0. Bafilomycin completely blocked endosome acidification, indicating that H+ transport is mediated by a vacuolar H(+)-adenosinetriphosphatase. Bafilomycin had no effect on transepithelial water flow in bladders repetitively stimulated by vasopressin. These findings, as well as the work of other investigators, suggest that if WC recycling occurs, it is not dependent on acidification of the endosomal compartment. Acidification of vasopressin-induced endosomes most likely represents a terminal event in the endocytic pathway.

Acids↗