The proton microprobe: a powerful tool for nondestructive trace element analysis.

A proton microprobe capable of focusing proton beams with energies up to 6 million electron volts to a spot size of 2 x 2 square micrometers has been used for chemical analysis of small grains of minerals in lunar samples by proton-induced x-ray emission. The proton microprobe is preferable to the electron microprobe for analyzing trace elements whose concentrations are below the detection limit of the latter and for analyzing objects with numerous major and trace elements with a wide range of atomic numbers. Application of the proton microprobe to biological samples is feasible.

Renin distribution in the rabbit renal microvasculature.

Immunocytochemical studies have shown that renin, which is normally located in the juxtaglomerular afferent arteriole, may also be found farther upstream toward the interlobular artery during chronic stimulation of the renin-angiotensin system. We assessed the renin distribution along the renal microvasculature using both quantitative analysis and immunocytochemistry in rabbits that received a normal sodium diet (0.48% NaCl), a low sodium diet (0.04% NaCl), or enalapril (1 mg/kg/day) for 4 weeks. From the outer cortex we microdissected 1) the proximal portion of the afferent arteriole (p-AF) extending from the interlobular artery to a point 50 microns from the glomerulus, 2) the distal 50 microns including its intact terminus (d-AF), and 3) the glomerulus without the vascular pole (GL) and measured their renin content. In controls, renin was 0.3 +/- 0.2, 27.0 +/- 5.2, and 2.8 +/- 0.5 ng angiotensin I/hr/arteriole (or GL) in the p-AF, d-AF, and GL, respectively. The low sodium diet and enalapril increased renin in the d-AF (53.1 +/- 6.9 and 68.4 +/- 8.1, respectively) but not in the GL (3.3 +/- 1.0 and 3.6 +/- 0.7). In the p-AF, both caused a small increase (delta = 1.5); however, this increase was minuscule compared with the large increase in the d-AF (delta = 41).(ABSTRACT TRUNCATED AT 250 WORDS)

Digital video-imaging of leukocyte migration in the iris: intravital microscopy in a physiological model during the onset of endotoxin-induced uveitis.

The process of inflammation is accompanied by an alteration of leukocyte-endothelial dynamics. Reciprocal changes in the endothelium and the white cell permit the leukocyte to relinquish its normal free-flowing state in order to roll, arrest, and emigrate through the endothelium. Although intravital microscopy is an established method to observe this process, the eye has been under-utilized for this purpose. Iris vasculature can be videophotographed without the artifact of trauma. We used rhodamine 6G in vivo staining of leukocytes from BALB/c mice in a model of inflammation induced by intravitreally injected endotoxin. Digital video technology was used to record observations at baseline, 2 h, and 4 h after the endotoxin injection. Off-line analysis of microhemodynamic parameters established that the percentage of venules exhibiting rolling increased significantly from 4% at baseline to 34% at 2 h and 82% at 4 h after endotoxin injection. We found a marked increase in leukocyte arrest within 4 h (601+/-119 cells per mm(2) vs. 2+/-1 cells per mm(2) in control animals). Although shear stress differs minimally between iris arterioles and venules, both rolling and arrest occurred preferentially in venules indicating that shear stress is not the dominant factor for determining cell adhesion. Compared to previous reports on intravital microscopy, our methodology includes refinements or advantages in visualizing cells that have transmigrated as well as the avoidance of surgical trauma. The resolution and quantifiable nature of this technique are such that the methodology can be applied to repetitive observation of leukocyte-endothelial dynamics during an immune response.

In vivo significance of ICAM-1--dependent leukocyte adhesion in early corneal angiogenesis.

PURPOSE: Numerous investigations have stressed the significance of leukocytes in early angiogenesis. Leukocytes invade the cornea, and the location of their extravasation corresponds to the site of vessel ingrowth. The interactions between leukocytes and vascular endothelium are mediated by various proteins, including adhesion molecules such as intercellular adhesion molecule-1 (ICAM-1). In this study, the role of ICAM-1 during early corneal angiogenesis was evaluated in vivo. METHODS: Corneal neovascularization was induced in New Zealand White rabbits by use of intrastromal pellets containing 750 ng vascular endothelial growth factor (VEGF). The fluorescent dye rhodamine 6G was used to stain leukocytes in vivo. Leukocyte adhesion and vessel growth were quantified in vivo by high-resolution fluorescence angiography. To inhibit ICAM-1 interactions a microemulsion containing anti-ICAM-1 antibody was applied topically. RESULTS: Limbal vessels showed increased leukocyte adhesion 24 hours after pellet implantation: The number of rolling and sticking leukocytes was significantly increased compared with the number in control animals (P < 0.01). Treatment with anti-ICAM-1 antibody resulted in reduced leukocyte sticking and increased leukocyte rolling. The area covered by new blood vessels was significantly diminished in eyes treated with anti-ICAM-1 (P < 0.05). CONCLUSIONS: The results support the hypothesis that ICAM-1-mediated leukocyte adhesion is a key event in early angiogenesis. This model may serve for investigation of the significance of adhesion molecules by in vivo observation and quantification.

Regulated expression of pancreatic renin-angiotensin system in experimental pancreatitis.

Our previous studies have provided evidence for the existence of an intrinsic renin-angiotensin system (RAS) in the rat pancreas, which may play a role in the regulation of pancreatic microcirculation and ductal secretion. Such a pancreatic RAS has recently shown to be activated by chronic hypoxia. The activation of a local RAS in the pancreas by chronic hypoxia and its significance of changes may be important for the physiological and pathophysiological aspects of the pancreas. In the present study, the regulation of experimentally induced acute pancreatitis on the expression of local RAS in the pancreas was investigated using Western blot, semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemical approaches. Results from Western blot demonstrated that experimentally induced pancreatitis caused significantly increased expression of the pancreatic RAS component proteins. In keeping with the protein level, RT-PCR analysis also revealed the enhanced expression of pancreatic RAS genes, notably the angiotensinogen in experimental pancreatitis. Immunohistochemical results further demonstrated that increased immunoreactivity for RAS in experimental pancreatitis was predominantly localized to the endothelia and epithelia of pancreatic vasculature and ductal system respectively. The data indicate that experimental pancreatitis may elicit activation of a local RAS in the pancreas. Such an activation of pancreatic RAS and its significance of differential changes in individual RAS components could play a role in the pathophysiology of acute pancreatitis

Epithelioid cells: membrane potential changes induced by substances influencing renin secretion.

Microelectrode recordings were performed in renin-containing epithelioid (JG) and vascular smooth muscle (VSM) cells of the afferent arteriole in the isolated hydronephrotic mouse kidney. Both cell types had a membrane potential of about -75 mV and exhibited small, spontaneous depolarizing transients, probably resulting from random transmitter release by sympathetic axon terminals. Substances depressing renin secretion, such as angiotensin II, arginine-vasopressin, and alpha 1-adrenergic agents reversibly depolarized both JG and VSM cells. On a molar basis, the action of angiotensin II was strongest. Stimulators of renin release, e.g. isoproterenol, histamine, and prostaglandin E2 did not influence the membrane potential of both cell types. VIP and NPY, possible co-transmitters of norepinephrine, as well as AP II, were also without effect. It is proposed that suppression of renin secretion from JG cells is mediated by depolarization and Ca2+ influx, whereas stimulation is triggered independently from membrane potential changes, e.g. by adenylate cyclase activation.

Activation of local renin-angiotensin system by chronic hypoxia in rat pancreas.

Previous studies have provided evidence that several key elements of renin-angiotensin system (RAS) are present in the rat pancreas, notably angiotensinogen, which is mandatory for intracellular generation of physiologically active angiotensin II. The data support the existence of an intrinsic RAS, which may be important for pancreatic blood flow and ductal anion secretion. In the present study, the effect of chronic hypoxia on the expression of RAS components, particularly at the levels of its precursor angiotensinogen and its receptor subtypes AT(1) and AT(2), were investigated in the rat pancreas. Results from western blot and semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) analyses unequivocally showed that chronic hypoxia caused a marked increase in angiotensinogen both at the protein and gene levels when compared with that in the normoxic pancreas. However, results from RT-PCR showed that there was a differential effect of chronic hypoxia on the expression of AT(1) and AT(2) receptor subtypes, which exhibited subtype-specific changes in gene expression. For AT(1), chronic hypoxia did not cause a significant change in mRNA expression for AT(1a) but a significant increase in mRNA expression for AT(1b). For AT(2), chronic hypoxia caused a marked increase in its mRNA expression. The increased expression of RAS component genes by chronic hypoxia and its significance of changes may be important for physiological and pathophysiological aspects of the pancreas.

Plasticity of postganglionic sympathetic neurons in the rat superior cervical ganglion after axotomy.

The neuropeptides galanin (GAL) and vasoactive intestinal polypeptide (VIP) are upregulated in spinal and vagal sensory as well as in cranial motor neurons after axonal transection. In this study an increase of both peptides is demonstrated in axotomized principal ganglionic neurons (PGN) of the rat sympathetic superior cervical ganglion by use of double-labeling immunofluorescence. Compared to control ganglia that do not contain more than 1% GAL- or VIP-positive cells, about 26% of all PGN exhibit GAL immunoreactivity by day 1 after transection of the major postganglionic branches. The proportion of immunoreactive neurons reaches its maximum after 30 days (40%) and decreases to about 27% within the second month after axotomy. The percentage of VIP-positive neurons is much lower than for GAL: 2% of the PGN exhibit VIP immunoreactivity at day 1 and about 7% are observed 30 and 60 days after axotomy. In order to further characterize newly GAL- and VIP-positive PGN, their cell diameters were determined 12 days after axotomy. Compared to the mean overall neuron diameter of 24.8 microns, GAL-immunoreactive neurons are predominantly of small and intermediate size (22.2 microns), whereas VIP occurs mainly in larger neurons (26.1 microns). Besides cell bodies, many intraganglionic nerve fibers stain positive for GAL or VIP, particularly at day 6. Most likely, these fibers represent axons, as indicated by the absence of MAP2, a cytoskeletal protein found in neuronal somata and dendrites. They establish direct membrane contacts with postganglionic perikarya, as revealed by pre-embedding immuno-electron microscopy. Some cell bodies and fibers contain both peptides. Colocalization of GAL or VIP with tyrosine hydroxylase (TH), the rate-limiting enzyme of catecholamine synthesis, reveals a reduced immunoreactivity for TH in intensely GAL- or VIP-positive cells, and vice versa at day 6. However, no difference in staining intensity for VIP or GAL, and TH, is observed after 30 and 60 days. Possible implications of GAL and VIP for peripheral nerve regeneration and their regulation by target-derived factors are discussed.

Prostaglandin F2 alpha modulates responses of single cultured mesangial cell to arginine vasopressin.

The role of prostaglandin F2 alpha (PGF2 alpha) interaction with arginine vasopressin (AVP) in modulating intracellular Ca2+ homeostasis was studied. Examinations were done on single cultured mesangial cells loaded with fura-2. Pretreatment of cultured mesangial cells during 1 min with different concentrations of PGF2 alpha (10(-5)-10(-8) M) caused a significant prolongation of [Ca2+]i transients after subsequent AVP applications. The observed effect of a prolonged sustained phase was not influenced by the temporal sequence of AVP stimulation after preincubation with PGF2 alpha: the signal was modulated in nearly the same way after 5 min delay as after nearly simultaneous application of AVP and PGF2 alpha. Measurements in Ca(2+)-free medium showed that the prolonged sustained phase of [Ca2+]i transients after AVP applications in cells pretreated with PGF2 alpha was mostly due to Ca2+ release from intracellular store(s). Pretreatment of the cells with PGF2 alpha also greatly enhanced the % of AVP responsive cells. Even at concentrations of AVP as low as 10(-10) M about 30% of cells pretreated with PGF2 alpha responded with the fast [Ca2+]i rise. Thus, present studies showed that PGF2 alpha specifically modulates [Ca2+]i transients after AVP stimulation and enhances sensitivity of mesangial cells to AVP. The results help to identify PGF2 alpha participation in the cellular regulatory mechanisms of microcirculation and filtration in the kidney.

A piezotranslator with variable movement pattern: experiences with the penetration of very small cells.

Successful recording of intracellular potentials strongly depends on the quality of the impalement of the cells by microelectrodes. A substantial improvement of the penetration process could be obtained by using a piezotranslator which accurately controls the forward and backward movement of the electrode tip. The relevant movement amplitudes and velocities can be adjusted independently. The described piezotranslator was used in experiments with cultured cells of the glomerular mesangium of rat kidney, forming a flat monolayer 1-3 micrometers in height. Many successful impalements and long-term, stable recordings demonstrate the usefulness of the translator.

The renin-angiotensin system: from the renal basis to an organ-specific subsystem in the pancreas.

Not only is the renin angiotensin system or its components found morphologically in many organs, it also exerts many different regulatory functions such as contributing to systemic homeostasis as well as to organ-specific regulation. The presence of the components of the renin angiotensin system in the pancreas was discovered only a few years ago. Physiological and pathophysiological stimuli were able to modify, in part, the gene expression and the occurrence of some of these components. Because of the important clinical significance of pancreatic diseases such as pancreatitis, research should follow every traces of the renin angiotensin system in the pancreas: impairment of microcirculation via hypoxia mediated up-regulation with the subsequent further deterioration of the oxygen supply seems to be the most obvious mechanism. There are many possible approaches to a better understanding of problems that are associated with diseases such as different kinds of pancreatitis; basic studies in animal models are oriented toward microcirculation, cellular function and the time course of modified gene expression after stimuli such as hypoxia; a clinical approach must reevaluate different correlations between clinical parameters of hypertension and those of pancreatic diseases.

The renin-angiotensin system in cats with chronic renal failure.

The kidneys of eight male and two female cats with subacute (clinical illness 1-3 months) to chronic (clinical illness > 3 months) renal failure were examined histopathologically, electron microscopically and immunohistochemically. Semiquantitative morphometric data, obtained by measurement of the reninpositive portion of the afferent arteriole (RPP) and evaluation of the juxtaglomerular index (JGI), were compared with data from three healthy control cats. On the basis of the morphometric data, the animals with renal failure could be classified in three groups showing either a stimulated (group A), an unaltered (group B) or an inhibited (group C) renin-angiotensin system. In the three group A cats the JGI and RPP were increased (45.5 +/- 3.5%; 130 microns); in the four group B cats these values were comparable with those of the controls; in the three group C animals the JGI was decreased but the RPP was unaltered (11.7% +/- 3.2%; 56 microns). The increase in kidney renin in animals affected by chronic renal failure (CRF) may have been due to a volume depletion. Prolonged CRF seemed to result in increasing hypertrophy of renal blood vessels, leading to renal hypoxia and increasing preglomerular resistance. Reduced kidney renin status may have been caused by inhibition of renin synthesis in prolonged CRF as a result of renal ischaemia.

Morphology of electrophysiologically identified baroreceptor afferents and second order neurones in the brainstem of the cat.

Baroreceptor afferent fibres and second order baroreceptor neurones were identified by their discharge pattern and were intracellularly injected with horseradish peroxidase. Three afferent fibres and three second order neurones were reconstructed by camera lucida drawings from serial sections of the brainstem. The afferent fibres were classified as A delta-fibres and had terminal arborizations with synaptic boutons in the dorsomedial region of the nuclei of the solitary tract (TS). The afferent fibres had additional collaterals with a medial projection to the commissural nucleus and in a direction lateral to the TS. The terminals of these collaterals could not be demonstrated. The second order neurones were located in the same dorsomedial region as the synaptic boutons of the afferent fibres. Neurones were small and spindle-shaped with two primary dendrites: one dendrite projected cranially along the medial border of the TS, and the second one projected caudally and medially into the commissural nucleus. The unmyalinated axons of these neurones could be traced over a distance of 1 mm. In only one neurone could an axon collateral be detected. The axons projected dorsally around the TS in a ventrolateral direction beyond the boundaries of the nuclei of the TS. The axon collateral projected in the medial direction into the commissural nucleus. In no case were axon terminals demonstrated.

Functional outcome after restorative proctocolectomy in pigs: comparing a novel transverse ileal pouch to the J-pouch and straight ileoanal anastomosis.

BACKGROUND: Restorative proctocolectomy followed by an ileoanal J-pouch procedure is the therapy of choice for patients with familial adenomatous polyposis and ulcerative colitis. After low anterior rectal resection, the authors have reported on a novel, less complex pouch configuration, a transverse coloplasty pouch. The aim of the present work was to apply this new design to the ileal pouch construction, to evaluate feasibility, and to measure functional results in comparison with the J-pouch and the straight ileoanal anastomosis using the pig as an animal model. METHODS: Twenty-three pigs underwent restorative proctocolectomy followed by reconstruction with straight ileoanal anastomosis (IAA; n = 5), J-pouch (n = 7), and a transverse ileal pouch (TIP; n = 11). Pigs were followed for 6 days postoperatively. Peristaltic function was assessed by manometry proximal to the pouch, in the reservoir, and at the level of the ileoanal anastomosis. Functional outcome was monitored by semiquantitative assessment of the general condition of the animals, postoperative feeding habits, and stool frequency and consistency. A Fourier analysis was performed in order to compare peristalsis in the ileal reservoirs. The reservoir volume was measured in situ by triple contrast computed tomography scan with 3D reconstruction. RESULTS: Seventeen animals survived for 1 week. There was no difference in the general condition or the feeding habits of the groups. A significant number of pigs with the TIP pouch (7/10) had semisolid or formed stools as opposed to liquid stools after J-pouch (6/6) and IAA (4/5; p = 0.01). TIP animals had a lower stool frequency (3.2 +/- 1.14 per day) on day 6 after the operation than pigs with J-pouch, 5.33 +/- 1,03, and IAA, 4.6 +/- 1.82 (p = 0.0036). The in situ volume of the pouches did not differ significantly. The Fourier analysis demonstrated a disruption of peristalsis by the J-pouch and the TIP reconstruction but not after IAA. CONCLUSION: The function of ileoanal reservoirs after proctocolectomy may result from the disruption of properistaltic waves after pouch formation. The mechanism of peristalsis disruption is independent of the in situ volume of the pouch.

The mesangial cell culture: a tool for the study of the electrophysiological and pharmacological properties of the glomerular mesangial cell.

Cultured rat glomerular mesangial cells (MC) were evaluated as a tool for reliable electrophysiological measurements as well as for fluorimetric determinations of intracellular Ca++. They had a resting potential similar to that observed in cultured vascular smooth muscle cells (VSMCs), in VSMCs of mouse kidney arterioles, or in glomerular--presumably mesangial--cells of kidney slices. The comparison with the other cell types was carried out in order to look for features distinguishing them from these cells, e.g., active and passive electrical membrane properties or electrical membrane responses to vasoactive pharmacological agents. In MCs, as well as in the other cell types, the average membrane potential was approx. -50 mV. The vasoconstrictor peptides angiotensin II (ANG II) and arginine-vasopressin (AVP) caused depolarizations that could be blocked by the respective specific inhibitors of these compounds. The agonist-induced depolarizations have to be attributed, at least in part, to a Ca++ inward current. Norepinephrine, if any, had only a weak action upon MCs, whereas isoproterenol either did not influence the membrane potential or hyperpolarized the cells. Other substances tested, which had no influences upon the membrane potential, were neuropeptide Y and atriopeptin 3. As to their resting electrical properties and their responses to pharmacological agents, cultured mesangial cells did not differ from glomerular, i.e., most probably mesangial, cells in the kidney slice. The difference between mesangial cells and VSMCs consists in their reaction to noradrenaline. Whereas VSMCs respond with a marked depolarization, the noradrenaline effect upon MCs in culture and in the kidney slice is either absent or very weak. Repeated passage of the cells (more than six passages) led to a gradual loss of their responsiveness to the agonists, indicating reduced receptor expression which may be interpreted as dedifferentiation. This held for both cultured MCs and VSMCs. Fluorimetric measurements using the Ca++-specific indicators quin-2 and fura-2 were performed with a purpose-developed, ultrasensitive photon-counting microspectrofluorimeter. Individual MCs as well as isolated glomeruli responded to the vasoconstrictors ANG II and AVP with an increase in Ca++-dependent fluorescence indicating that these agents indeed depolarize the cells partly via a Ca++ influx and increase cytosolic free Ca++.(ABSTRACT TRUNCATED AT 400 WORDS)

Influence of pulsatile perfusion upon renin release from the isolated perfused rat kidney.

It is well established that renin release from the juxtaglomerular epithelioid cells in the media of the afferent arteriole strongly depends on the mean renal perfusion pressure, whereas a possible influence of the pulsation of blood pressure on renin release has only occasionally been investigated, and the results are contradictory. Such an influence on renin release cannot be excluded because pulsation is known to modulate arterial baroreceptors and vascular tone in some resistance vessels. In the isolated perfused rat kidney, we found a pulsation amplitude-dependent inhibition of renin release that could be blocked either by vasodilatation or by calcium channel blockade. The inhibition occurred at perfusion pressures between 85 and 125 mm Hg. The underlying pulsation pressure-sensitive mechanism has to be ascribed integrating properties, because a constant-flow pressure rise to the "systolic" value of pulsatile perfusion resulted in virtually the same inhibition of renin release. Moreover, a reduced urine flow during pulsatile perfusion provides evidence for preglomerular constriction under these conditions. It is concluded that, besides pathological changes of renal perfusion pressure, variations of the pulse amplitudes, e.g. resulting from renal artery stenosis or atherosclerosis, may also influence renin release and contribute to renovascular hypertension.

Coexistence of renin and cathepsin B in epithelioid cell secretory granules.

Mature juxtaglomerular epithelioid cell secretory granules of the rat exhibit both renin- and cathepsin B-like immunoreactivity. On the basis of the coexistence with renin at a pH which, according to previous experiments, is probably in the range of that in lysosomes, cathepsin B is suggested to be involved in the activation of renin prior to secretion.

Junctional transmission in renin-containing and smooth muscle cells of the afferent arteriole.

Intracellular recordings were done in renin-containing juxtaglomerular (JG) and vascular smooth muscle (VSM) cells of the mouse kidney afferent arteriole. Both cell types exhibited a membrane potential around -75 mV and spontaneous depolarizing transients resembling spontaneous excitatory junction potentials (SEJPs) in the arterioles of other organs. The amplitude distribution of these randomly occurring transients was skewed in both cell types with a modal value of 1.2-1.9 mV. Activation of presumably postjunctional alpha 1-, P2-, ANG II- and AVP-receptors depolarized JG and VSM cells. Application of the P1-purinoceptor agonist 2-chloroadenosine strongly increased frequency and amplitude of the SEJP-like events, whereas these transients were abolished by the P1-purinoceptor antagonist 8-phenyltheophylline, both substances presumably acting on prejunctional receptors. The SEJP-like events were completely depressed by reserpine treatment, but not abolished by alpha 1-, alpha 2-, and P2-antagonists. At present, it cannot be decided, whether norepinephrine is the sole transmitter in the afferent arteriole, acting on specialized junctional adrenoceptors with the P2-purinoceptors being irrelevant for junctional transmission, or whether both substances are co-transmitters. Except norepinephrine and ATP, all other transmitter candidates tested were ruled out for various reasons.