Electron microscope observations on form changes in photoreceptor outer segments and their saccules in response to osmotic stress.

Isolated retinas of rats or frogs were incubated in various salt or sucrose solutions over a wide range of osmolarities and then fixed in 1% osmium tetroxide solutions of matching osmolarities. Light and electron microscope observations were concentrated on the outer segments of the intact photoreceptors. These became globular and of increasing size with increasing hypoosmolarity and irregularly linear and condensed in hyperosmotic solutions. Isoosmotic incubations of rat retinas in solutions containing potassium as the only cation also produced swelling of the outer segments when chloride or acetate was present; but swelling was less when the cation was sodium and it was not seen with either cation when the anion was methylsulfate. The effects of various metabolic and membrane poisons are also reported. The behavior of the saccules within the outer segments was equivocal. While there was a tendency toward more saccules with wider lumina with hypoosmolarity, most of the saccules were not swollen. Surprisingly, intrasaccular space was consistently enlarged in rat retinas exposed to hyperosmotic sucrose but not to salt. The saccules or their derivatives within swollen outer segments tend to maintain their intersaccule spacing and approximation to the cell membrane. It was also noted that the ciliary connectives resist swelling and that retinal Müller cells swell readily.

New evidence supporting the linkage to extracellular space of outer segment saccules of frog cones but not rods.

Previous electron microscopic examinations of outer segments of photoreceptors suggest that many flattened saccules of cones are continuous with the cell membrane and that their lumina connect with the extracellular compartment but that most saccules in rods appear to lack these connections. The saccules probably contain photolabile pigment, and certain potentials appear to result from dipole formation during pigment bleaching. The detection of dipoles from rod saccules may require that the lumina of rod saccules connect with extracellular space, and questions have been raised whether the interpretation of micrographs is correct or the isolation of rod saccules is the result of artifact. Accordingly, lanthanum and barium precipitates were produced near fixed and unfixed frog photoreceptors. Lanthanum precipitates appeared to infiltrate the saccules of fixed cones and the few surviving cones exposed prior to fixation, but no rod saccules were infiltrated except occasional, most basal saccules or saccules within narrow zones of probable damage. Barium precipitates did not infiltrate saccules of either variety of unfixed photoreceptor, but they did occasionally infiltrate around the saccules at points of damage in rod outer segments. The results thus support the view of the patency of saccules of frog cones and are consistent with, but do not prove, the isolation of saccules of frog rods.

Freeze-fracture studies of photoreceptor membranes: new observations bearing upon the distribution of cholesterol.

We performed electron microscopy of replicas from freeze-fractured retinas exposed during or after fixation to the cholesterol-binding antibiotic, filipin. We observed characteristic filipin-induced perturbations throughout the disk and plasma membranes of retinal rod outer segments of various species. It is evident that a prolonged exposure to filipin in fixative enhances rather than reduces presumptive cholesterol detection in the vertebrate photoreceptor cell. In agreement with the pattern seen in our previous study (Andrews, L.D., and A. I. Cohen, 1979, J. Cell Biol., 81:215-228), filipin-binding in membranes exhibiting particle-free patches seemed largely confined to these patches. Favorably fractured photoreceptors exhibited marked filipin-binding in apical inner segment plasma membrane topologically confluent with and proximate to the outer segment plasma membrane, which was comparatively free of filipin binding. A possible boundary between these differing membrane domains was suggested in a number of replicas exhibiting lower filipin binding to the apical plasma membrane of the inner segment in the area surrounding the cilium. This area contains a structure (Andrews, L. D., 1982, Freeze-fracture studies of vertebrate photoreceptors, In Structure of the Eye, J. G. Hollyfield and E. Acosta Vidrio, editors, Elsevier/North-Holland, New York, 11-23) that resembles the active zones of the nerve terminals for the frog neuromuscular junction. These observations lead us to hypothesize that these structures may function to direct vesicle fusion to occur near them, in a domain of membrane more closely resembling outer than inner segment plasma membrane. The above evidence supports the views that (a) all disk membranes contain cholesterol, but the particle-free patches present in some disks trap cholesterol from contiguous particulate membrane regions; (b) contiguous inner and outer segment membranes may greatly differ in cholesterol content; and (c) the suggested higher cholesterol in the inner segment than in the outer segment plasma membrane may help direct newly inserted photopigment molecules to the outer segment.

Freeze-fracture evidence for the presence of cholesterol in particle-free patches of basal disks and the plasma membrane of retinal rod outer segments of mice and frogs.

The freeze-fracture technique was used to examine the membranes of the photoreceptors of mice and frogs. Particle-free patches were found in the plasma membrane and basal disk membranes of the outer segments of both mice and frogs housed at room temperature, but not in frogs kept in a cold room. These patches were shown not to be artifacts of cryoprotection or fixation, and they persisted when fresh isolated outer segments were frozen by an ultrarapid method. They were also found to persist in mouse rods when retinas were incubated and subsequently fixed at temperatures up to 80 degrees C. Cholesterol was implicated as a significant component of the patches by the observation that, in the outer segments, pits, induced by treatment with the sterol-specific polyene antibiotic filipin, were present in and confined to the particle-free patches. That these lesions are not inherently limited to particle-free membrane areas was evident in the apical plasma membrane of the photoreceptor inner segments, where particles and pits were intermixed. Treatment with saponin, a surface-active agent which specifically complexes cholesterol, resulted in the disappearance of the particle-free patches. Patches were found in basal disks of both mouse and frog rods but not in older disks nearer the pigment epithelium, which indicates that changes occur in the composition of disk membranes and/or in the molecular ordering of their protein and lipid components during the early phase of their transit from the base towards the apex of the outer segment.

Light-induced losses and dark recovery rates of guanosine 3',5'-cyclic monophosphate in rod outer segments of intact amphibian photoreceptors.

We used an apparatus in which pieces of dark-adapted amphibian retinas (Rana pipiens, Bufo marinus) obtained under infrared illumination were exposed to precise intervals of 500-nm illuminations, and then frozen by contact of their outer segment surface with a liquid helium-cooled copper mirror. Sections of the frozen outer segment layer were obtained in a cryostat and then assayed for total extractable cyclic 3',5'-guanosine monophosphate (cGMP). Significant losses of cGMP with respect to the dark level were evident as early as 60 ms after light onset. With dim subsecond illuminations these losses were surprisingly large, which suggests a previously underestimated magnification in the cGMP cascade, or a transient substantial inhibition of guanylate cyclase activity in combination with increased cyclic GMP phosphodiesterase activity. Within the subsecond period, significant losses that were proportional to light intensity (2-log-unit range) and duration (60-550 ms) were generally not evident. However, losses significantly proportional to these factors became evident with durations of 1 s or longer. When pieces of retina were first illuminated (10 or 60 ms), then held in darkness for increasing periods before freezing, we observed a continuous loss of cGMP during the early postillumination dark period, followed by a recovery of the total cGMP level. The times for recovery to the preillumination level appear to be significantly longer than times reported for the recovery of the photoreceptor membrane potential after similar light exposures.

Calcium and cyclic nucleotide regulation in incubated mouse retinas.

When retinas from dark-adapted C57BL/6 mice were incubated in the dark for 5 min at 37 degrees C in Earle's medium, they contained 80-120 pmol/mg protein of cGMP and about 13 pmol/mg protein of cAMP. When the incubation in darkness was in calcium-deficient Earle's medium with 3 mM EGTA, a 10-20 fold increase occurred in the cGMP level, peaking at 2-3 min, but no change occurred in cAMP. This elevated level fell in 3 min to normal dark levels on return to normal Earle's medium, but was still about three times that of control levels after 15 min in EGTA-containing solution. Bright light after 2 min of dark incubation of dark-adapted retinas resulted in a 40-50% fall in cGMP, and bright light sharply reduced the elevated dark cGMP level of retinas in calcium-deficient media with 3 mM EDTA. However, no depression of normal dark levels of cGMP has thus far been obtained by increasing external calcium levels, even in the presence of the ionophore A23187. All the above phenomena involving dark cGMP levels and calcium are similar in Earle's medium with 100 mM of K+ substituted for Na+. Congenic rodless (rd/rd) mouse retinas have less than 5% of control cGMP and show only traces of calcium sensitivity. Thus, the above phenomena in controls are likely to be largely occurring in rods. The data suggest a dependency of the dark cGMP level on the calcium level, but that the light-induced fall in cGMP may largely be calcium insensitive.

Reduced acetylation of procainamide by para-aminobenzoic acid.

Acetylation is the major route of metabolism of many drugs including the antiarrhythmic agent procainamide. Coadministration of para-aminobenzoic acid was observed to decrease the biotransformation of procainamide to N-acetylprocainamide in a patient with rapid acetylation kinetics. In view of the distinct antiarrhythmic and toxic properties of procainamide and N-acetylprocainamide, the observed drug interference may have great clinical relevance in long-term oral antiarrhythmic therapy and in instances where other drugs converge for acetylation.

Some cytological and initial biochemical observations on photoreceptors in retinas of rds mice.

As a control for biochemical studies in progress, an ultrastructural study has been carried out on the deteriorating, 21-day photoreceptors of the 020/Cpb strain of mice, homozygous for retinal degeneration, slow (rds). At 21 postnatal days, outer segments were essentially lacking, but cilia erupting from the inner segments were common. A low percentage of cilia bore small cytoplasmic masses containing a few layered membranes, and rare inner segments possessed spherical aggregations of multilayered membranes. Pigment epithelial cells also possessed membranous aggregations in presumed phagosomes. While other parts of photoreceptors possessed the usual organelles of normal rods, inner segments were reduced in volume, and the layer of photoreceptor synaptic terminals was thinner. Mutant 21-day retinas possessed about two-thirds of the protein of normal 21-day retinas but 50% more protein than "rodless" (rd/rd) 21-day retinas. Surprisingly, while dark-adapted rds retinas possessed markedly lower levels of cyclic GMP as compared to controls, light-adaptation significantly reduced cyclic GMP and cyclic AMP levels, and biochemical data point to persistent light-modulated cyclic nucleotide levels in the photoreceptors.

Adenosine: autoradiographic localization and electrophysiologic effects in the cat retina.

Autoradiography with 3H-adenosine was used to localize cells that accumulate adenosine in the cat retina. Electrophysiologic effects elicited by adenosine on DC-electroretinograms (ERG) and optic nerve responses (ONR) were studied in isolated, arterially perfused cat eyes. Subpopulations of cells localized in the ganglion cell layer and inner nuclear layer showed clear labeling for adenosine. This purine nucleoside enhanced the ERG b-wave and the standing potential; depressed the light peak; and markedly depressed the ONR, in which it reduced the amplitudes of the ON-, plateau-, and OFF-components. A vasodilatory action of adenosine was documented by an increase in perfusion flow rate. Our data suggest that adenosine in cat retina has complex modulatory effects, involving the retinal pigment epithelium, neuronal structures, blood vessels, and probably glial cells.

Pharmacokinetics of captopril in healthy subjects and in patients with cardiovascular diseases.

Captopril, the first orally active inhibitor of angiotensin-converting enzyme, is used widely in the treatment of hypertension and congestive heart failure. The pharmacokinetics of this agent have been studied extensively in healthy subjects and in patients with hypertension, congestive heart failure, and chronic renal failure. Captopril contains a sulphydryl group and binds readily to albumin and other plasma proteins. The drug also forms mixed disulphides with endogenous thiol-containing compounds (cysteine, glutathione), as well as the disulphide dimer of the parent compound. These components in blood and urine are measured collectively as total captopril. Because of the reversibility of the formation of these inactive disulphides, total captopril may serve as a reservoir of the pharmacologically active moiety, and thus contribute to a duration of action longer than that predicted by blood concentrations of unchanged captopril. To measure free or unchanged captopril concentrations, a chemical stabiliser must be added to the biological samples to prevent the formation of captopril disulphides ex vivo. In healthy subjects given captopril intravenously, the body clearance of captopril and steady-state volume of distribution were about 0.7 L/h/kg and 0.8 L/kg, respectively. The elimination half-life of unchanged captopril was approximately 2 hours. The primary route of elimination of captopril is the kidney. The renal clearance of unchanged captopril exceeds the glomerular filtration rate, due to active tubular secretion of the drug. In healthy subjects, about 70 to 75% of an oral dose is absorbed and the bioavailability of captopril is approximately 65%. Peak blood concentrations are reached about 45 to 60 minutes after oral administration. The bioavailability of captopril is not altered by age or concomitant medications including diuretics, procainamide, allopurinol, cimetidine or digoxin. However, the co-administration of food or antacids, or probenecid with captopril has been shown to diminish the bioavailability of the latter and decrease its clearance, respectively. The decreased bioavailability of captopril when taken with meals does not significantly alter clinical responses to the drug. Over a wide range of oral (10 to 150 mg) and intravenous doses (2.5 to 10 mg) captopril had linear kinetics in healthy volunteers. In healthy subjects with normal renal function and patients with congestive heart failure given captopril 3 times daily, blood concentrations of total captopril accumulated, whereas those of unchanged captopril did not. Severe renal insufficiency was associated with an accumulation of both unchanged and total captopril.(ABSTRACT TRUNCATED AT 400 WORDS)

Importance of left atrial timing in the programming of dual-chamber pacemakers.

To determine the hemodynamic effect of different programmed atrioventricular (AV) delays and the importance of the actual timing of left atrial (LA) depolarization, 16 patients with dual-chamber pacemakers were studied and all were found to have an optimal programmed AV delay for cardiac function. However, randomly chosen AV delays of 150, 200 or 250 ms actually provided worse stroke volume than VVI pacing in 7 patients. The optimal programmed AV delay was variable between patients and was related to the interatrial conduction delay, measured as the time from right atrial pacing artifact to LA depolarization (mean 144 +/- 82 ms, range 70 to 380.) Patients with short interatrial delays (less than or equal to 90 ms) were served better by shorter programmed AV delays (150 ms), and patients with longer interatrial delays (greater than or equal to 120 ms) were served better by longer programmed AV delays (greater than or equal to 200 ms) (p less than 0.05). Furthermore, as pacing mode changed from dual-chamber sequential pacing (DVI) to atrial synchronous ventricular pacing (VDD), the LA to ventricular sequence increased from 6 +/- 81 ms to 137 +/- 50 ms (p less than 0.001). This change in the LA to ventricular sequence with mode change produced a significant decrease in stroke volume (p less than 0.05). Thus, the optimal programmed AV delay in patients with dual-chamber pacemakers is predicted by the relation of LA and ventricular activation. Because interatrial conduction delays vary widely, optimal programming requires knowledge of the LA to ventricular sequence.(ABSTRACT TRUNCATED AT 250 WORDS)

M-mode echocardiograms for determination of optimal left atrial timing in patients with dual chamber pacemakers.

To determine if the A wave of the mitral valve echocardiogram can be used as a marker for left atrial (LA) activity and assist in the programming of dual chamber pacemakers, 156 echocardiograms with the mitral A wave present were obtained from 23 patients with dual chamber pacemakers, all of whom had bipolar esophageal recordings of LA depolarization. Twelve of these patients also underwent hemodynamic study with cardiac function determined at 5 different pacemaker settings: ventricular demand pacing and dual chamber sequential pacing at 0 or 25, 150, 200 and 250 ms programming atrioventricular (AV) delay. The time delay from right atrial pacing artifact to onset and peak of mitral A wave was linearly related to the time from atrial pacing artifact to LA depolarization on the esophageal lead (p less than 0.001). As pacing mode changed from dual chamber sequential pacing (DVI) mode to atrial synchronous-ventricular pacing (VDD), the A wave came earlier relative to the ventricular pacing spike, linearly related to the LA to ventricular extension with mode change determined with the esophageal lead (r = 0.94, p less than 0.001). The time from atrial pacing to peak of A wave was shorter in patients whose optimal programmed AV delay was 150 ms compared with those whose optimal AV delay was 200 or 250 ms (p less than 0.02). At the optimal programmed delay for cardiac output, the peak of the A wave was an average of 13 +/- 36 ms after the ventricular pacing spike.(ABSTRACT TRUNCATED AT 250 WORDS)

Noninvasive electrophysiologic study using standard permanent pacemakers in patients with spontaneous sustained ventricular tachycardia or ventricular fibrillation.

Permanent pacemakers capable of noninvasive electrophysiologic testing were used to study and treat 26 patients with spontaneous sustained ventricular tachycardia (VT) or fibrillation (VF). One hundred nine episodes of sustained VT or VF were induced in these patients. In 8 patients spontaneous VT was reverted by noninvasive means. Drug changes based on noninvasive testing were made in 12 patients. In the 1- to 67-month follow-up period, drug therapy based on noninvasive electrophysiologic testing was predictive of outcome in patients with spontaneous arrhythmias. Thus, noninvasive electrophysiologic testing using permanent pacemakers is a useful method for studying and treating patients with recurrent sustained ventricular arrhythmias.

Serial electrocardiographic changes in idiopathic dilated cardiomyopathy confirmed at necropsy.

Serial electrocardiographic changes in necropsy-proven idiopathic dilated cardiomyopathy are evaluated and a method of predicting heart weight using QRS amplitudes is described. In 34 patients with multiple electrocardiograms (mean 3/patient) progressive prolongation of PR interval (0.18 +/- 0.03 to 0.21 +/- 0.03, p less than 0.001) and QRS duration (0.10 +/- 0.02 to 0.13 +/- 0.03, p less than 0.0001) was noted. Progressive conduction abnormalities were common (82%). QTc interval and QRS- and T-wave axes did not change. In 50 patients with electrocardiograms within 60 days of death, total 12-lead QRS and V1 through V6 QRS amplitude correlated better with heart weight (r = 0.51, p less than 0.0001 and r = 0.55, p less than 0.0001) than the Estes-Romhilt score did. The mean total 12-lead QRS amplitude was 138 mm with a mean of 106 for V1 through V6. In 31 patients cardiac mass index was calculated and showed significant correlation with 12-lead and V1 through V6 QRS amplitudes (r = 0.68, p less than 0.0001 and r = 0.75, p less than 0.0001, respectively). The QRS amplitudes remained constant during the illness. By using total 12-lead QRS or frontal plane QRS amplitude, heart weight can be predicted as early as 2 years before death. Use of body surface area and QRS amplitude criteria increases the accuracy of heart weight prediction. Thus, progressive electrocardiographic changes are common in patients with idiopathic dilated cardiomyopathy and QRS amplitude criteria are more accurate in the prediction of left ventricular hypertrophy than standard criteria.

Renal excretion of metolazone, a new diuretic.

1. Metolazone, a new diuretic, was found to be excreted by glomerular filtration and renal tubular secretion.2. The secretory mechanism was antagonized by probenecid but this did not affect the diuretic action of metolazone.

Allogeneic transplantation: a therapeutic option for myelofibrosis, chronic myelomonocytic leukemia and Philadelphia-negative/BCR-ABL-negative chronic myelogenous leukemia.

The role of allogeneic transplantation for myeloproliferative diseases other than chronic myeloid leukemia is not well established. In all, 20 patients with a median age of 51 years underwent allogeneic hematopoietic stem cell transplantation (HSCT) for myelofibrosis (n=5), chronic myelomonocytic leukemia (CMML) (n=8) and Philadelphia (Ph) chromosome-negative/BCR-ABL-negative chronic myeloid leukemia (CML) (n=7) in our institution. Patients who developed acute leukemia prior to HSCT were excluded from this analysis. A total of 15 patients received related and five patients received unrelated donor transplants. One patient failed to engraft. After a median follow-up of 17.5 months, actuarial survival at 2 years was 47% (95% CI 2%-67%), and disease-free survival 37% (95% CI 17-58%). Allogeneic transplantation may provide a therapeutic option for patients with myelofibrosis, CMML and Ph chromosome-negative/BCR-ABL-negative CML.

Regulation of cyclic AMP levels in mammalian retina: Effects of depolarizing agents and transmitters.

Cellular depolarization in brain results in a modulation of cAMP levels by releasing neurotransmitters having receptors linked via GTP-binding proteins to adenylate cyclase. In order to determine the transmitters regulating cAMP during cellular depolarization in mammalian retina, the modulation of cAMP by depolarizing media was investigated. Cyclic AMP levels in light adapted retinas increased following exposure to depolarizing media, but levels in dark adapted retinas remained unaltered. The depolarization-induced modulation of cAMP levels persisted in dystrophic retinas, suggesting that the response occurred in the inner retina. In microdissected discrete retinal layers from rabbit, levels of cAMP were increased following perfusion with depolarizing medium in the outer plexiform and inner nuclear layers, consistent with the observation seen with mouse retinas. To begin to identify transmitters released by cellular depolarization, a variety of transmitters and/or antagonists were included in the incubation medium. Haloperidol reduced the depolarization induced increase in cAMP levels by 25% in normal mouse retinas, and 75% in dystrophic retinas. Dopamine elevated cAMP levels in normal and dystrophic mouse retinas, and when combined with depolarizing medium, additive increases were observed. The effects of various neurotransmitters on retinal cAMP levels in the absence of any phosphodiesterase inhibitors were assessed, and both dopamine and norepinephrine were found to increase cAMP levels in normal and dystrophic retinas. Phentolamine antagonized the increase elicited by norepinephrine. When dopamine and norepinephrine were combined non-additive increases were observed. Serotonin, GABA, acetylcholine, histamine and adenosine had little or no significant effect on the retinal levels of cAMP in either normal or dystrophic mouse retinas. These results indicate that depolarizing media increase cAMP levels partially by releasing dopamine. The processes regulating cAMP levels in retina are both different and similar to those in brain.

Localization and roles of cyclic nucleotide systems in retina.

The distribution of cyclic GMP, cyclic AMP and related enzymes in the vertebrate retina, together with factors regulating their levels, are described. Photo-receptor cells in retinas from all species examined contain very high levels of cyclic GMP and high activities of both guanylate cyclase and cyclic GMP phosphodiesterase. In more proximal regions of the retina, cyclic GMP is found at concentrations similar to that of brain. Guanylate kinase and GDP kinase, enzymes involved in GMP metabolism, also have increased activities in photoreceptor cell layers although their pattern of distribution does not exactly parallel that of cyclic GMP. The concentration of cyclic AMP is fairly uniform throughout the retina and at a level similar to that found in other areas of the CNS. However adenylate cyclase has an uneven distribution with particularly high activity in the inner plexiform layer. Cyclic nucleotide levels in retina may be modified by several factors. Light decreases both cyclic nucleotides in rod-dominant retinas, although we have not observed similar changes in cone-dominant retinas. Anoxia or ischemia elevates cyclic AMP and decreases cyclic GMP, similar to other areas of CNS, while incubation of retina in Ca(++)- free media markedly increases cyclic GMP levels, an effect opposite that seen in brain tissue. Depolarization of retina with high K(+) causes a modest elevation of cyclic AMP but has no effect on cyclic GMP, which is also significantly different from the response in brain. Cyclic AMP levels in retina however, can be elevated by dopamine which is an effect similar to that in striatum. These data indicate that there are probably multiple cyclic GMP and cyclic AMP systems in retina, some of which may be unique to this tissue.

Transdiaphragmatic implantation of the automatic implantable cardioverter defibrillator.

A new surgical approach for implantation of the automatic implantable cardioverter defibrillator without thoracotomy was used in 12 patients, aged 46 to 72 years. Preimplantation arrhythmia was ventricular tachycardia in 7 patients and ventricular fibrillation in 5 patients. The mean ejection fraction was 19%. Six patients were at high risk for general anesthesia for a variety of medical problems, and 2 patients had had a previous cardiac operation. Epidural anesthesia was used in 8 patients without intubation. The surgical approach used a longitudinal epigastric extraperitoneal incision with access to the heart through an incision made in the central tendon of the diaphragm. Two patches and two epicardial sensing leads were placed in all patients. All patients but one could be defibrillated with 20 J or less. There was no operative mortality and minimal morbidity. There were two late deaths due to heart failure. Thus, the transdiaphragmatic approach provides an excellent exposure for automatic implantable cardioverter defibrillator implantation, avoids general anesthesia and thoracotomy, and can be used after a previous cardiac operation.

Interphotoreceptor contacts at the inner segment level in primate retinas.

Just sclerad to the external limiting membrane of the retina of the monkey (M. fasciculata), short processes from some foveolal and foveal cone inner segments made dense contacts with each other and at the same level of peripheral human retina similar rod-cone and rod-rod contacts were observed.