Serum-derived vitronectin influences the pericellular distribution of type 1 plasminogen activator inhibitor.

Bovine aortic endothelial cells (BAEs) were used as a model system to study the nature and origin of protein(s) in the extracellular matrix that bind to type 1 plasminogen activator inhibitor (PAI-1). Matrix samples were fractionated by SDS-PAGE and analyzed by PAI-1 ligand binding and by immunoblotting using antibodies to vitronectin (Vn). PAI-1 bound primarily to two Vn-related polypeptides of Mr 63,000 and 57,000, and both of these partially degraded polypeptides were present in the culture serum. Radiolabeling experiments failed to detect significant Vn biosynthesis by BAEs (less than 0.03% of total), or by human umbilical vein endothelial cells and HT 1080 cells. The binding of PAI-1 to Vn was relatively specific since direct binding studies failed to demonstrate significant interactions between PAI-1 and other matrix proteins (e.g., fibronectin, type IV collagen, laminin, or matrigel). Kinetic studies indicate that PAI-1 rapidly accumulates in the matrix when BAEs are plated on Vn, appearing in the conditioned medium only after a significant lag period (1-2 h). However, no PAI-1 was detected in the matrix when the cells were plated on fibronectin-coated dishes, and there was no lag period for PAI-1 accumulation in the medium. These results indicate that PAI-1 binds specifically to serum-derived Vn in the matrix, and suggest that the composition of both the matrix and serum itself may influence the pericellular distribution of this important inhibitor.

Elaboration of type b capsule by Haemophilus influenzae as a determinant of pathogenicity and impaired killing by trimethoprim-sulfamethoxazole.

In vitro, Haemophilus influenzae strains have two distinct patterns of susceptibility to trimethoprim-sulfamethoxazole (TMP/SMZ); strains with low minimum inhibitory concentration and high minimum bactericidal concentration (tolerant) and those with both low minimum inhibitory concentration and minimum bactericidal concentration (kill-sensitive). Tolerant H. influenzae strains were found to elaborate significantly more type b capsular polysaccharide, a linear polymer of ribosyl ribose phosphate (PRP), than kill-sensitive strains. Tolerant strains became susceptible to killing by TMP/SMZ when type b capsule was physically removed, but reacquired tolerance following growth and reversion to original (mucoid) phenotype. Susceptibility of wild (type a, b, c), isogenic (type b and untypable), and transformed (type b and d) strains indicated that elaboration of type b capsule was associated with TMP/SMZ tolerance. In a second series of studies, virulence of H. influenzae in the infant rat model was correlated with in vitro tolerance. Tolerant strains (13/13) caused systemic disease while none (0/7) of kill-sensitive strains were pathogenic. The efficacy of TMP/SMZ in the treatment of invasive infection was evaluated in rats with established bacteremia and meningitis. TMP/SMZ failed to eradicate H. influenzae b from the blood in 85% (17/20) or from the cerebrospinal fluid in 95% (19/20) of infected animals. Thus, in vitro tolerance correlated with therapeutic failure in vivo.

Effect of oxygen on the cytotoxicity and antitumor activity of etoposide.

The selective cytotoxicity of the epipodophyllotoxin etoposide toward normally oxygenated and hypoxic EMT6 mouse mammary tumor cells in culture was examined. Etoposide was much more toxic to normally oxygenated cells. The ratio (hypoxic to oxygenated) of drug concentrations producing 1 log of cell kill was approximately 30:1. Established FSa-11C fibrosarcomas of C3HeB/FeJ mice were treated with 10, 15, or 20 mg etoposide/kg body weight in a 6-day protocol. Fluosol-DA with or without breathing of carbogen (i.e., 95% O2-5% CO2) was added to the treatment program on days 1, 3, and 5. The combination of etoposide-Fluosol-DA-carbogen markedly enhanced tumor growth delay compared to the result with etoposide alone. The dose-modifying effect observed was 1.9 +/- 0.3. With the use of both single-dose and multiple-dose protocols for etoposide and Fluosol-DA with air or carbogen breathing, the survival of bone marrow cells was measured by colony formation in vitro (granulocyte-monocyte colony-forming units). Fluosol-DA and carbogen breathing did not increase the toxicity of etoposide to the bone marrow. Thus the enhancement in antitumor activity produced by the addition of Fluosol-DA and carbogen breathing to etoposide treatment was not accompanied by a concomitant increase in normal tissue toxicity and represents an increase in the therapeutic efficacy of etoposide.

Effect of various oxygenation conditions and fluosol-DA on cytotoxicity and antitumor activity of bleomycin in mice.

In an attempt to improve the antitumor efficacy of bleomycin, the effects of the oxygen-carrying emulsion Fluosol-DA and increased levels of inspired oxygen were tested in the mouse FSaIIC fibrosarcoma system. The dose-dependent cytotoxicity of bleomycin toward the FSaIIC cells in vitro was significantly decreased under hypoxic conditions, but it increased in a 95% O2-5% CO2 (carbogen) atmosphere as compared with the cytotoxicity of bleomycin in normally oxygenated cells. Investigations on the FSaIIC tumor in vivo also demonstrated that growth delays induced by bleomycin (10 mg/kg ip given on days 6, 10, 13, and 16) were significantly increased when one of the following treatments was given with each bleomycin injection: carbogen breathing for 2 hours (4.7 days), carbogen breathing for 6 hours (5.7 days), and breathing 3 atm of hyperbaric oxygen (6.3 days) versus normal air (3.3 days). When Fluosol-DA (12 mL/kg iv) was administered just before each bleomycin injection, the following growth delays were produced: 4.8 days with air breathing, 14.6 days with carbogen breathing for 2 hours, 14.9 days with carbogen breathing for 6 hours, and 19.7 days with breathing 100% O2 at 3 atm for 1 hour. Excision studies on the FSaIIC tumor also demonstrated that the cytotoxicity increased approximately fivefold when Fluosol-DA and carbogen breathing for 2 hours were combined with a single treatment with 10 mg of bleomycin/kg. In contrast, no measurable bone marrow toxicity was evident with this combined regimen. These results suggest that the use of Fluosol-DA plus carbogen breathing could add substantially to the clinical antitumor effects of bleomycin.

Modification of photodynamic therapy-induced hypoxia by fluosol-DA (20%) and carbogen breathing in mice.

The administration of a perfluorochemical emulsion and carbogen (95% O2, 5% CO2) breathing before photodynamic therapy (PDT) was studied to determine how increased levels of tumor oxygenation may affect PDT-induced tumor destruction. C3H/HeJ mice bearing the RIF tumor were given injections of 5 to 10 mg/kg of dihematoporphyrin ethers 24 h prior to treatment. Animals were given injections of 12 ml/kg of Fluosol-DA (20%) followed by carbogen breathing or 12 ml/kg of saline and air breathing (controls) 1 h before tumors were exposed to 135 J/cm2 of 630-nm light treatment. Changes in the hypoxic fraction of tumors, the time course for decreases in tumor cell clonogenicity, and tumor response were measured immediately and at various times after treatment. The administration of Fluosol-DA (20%) and carbogen breathing was found to delay the onset of PDT-induced hypoxia through the first hour posttreatment. Progressive tumor hypoxia was observed after 4 h posttreatment. The time period in which tumors remained well oxygenated coincided with observations of increased tumor cell survival. Decreases in tumor cell clonogenicity were observed only after tumor cells became hypoxic. These findings were consistent with the 24-h delay in complete tumor response in animals given Fluosol-DA (20%) and carbogen breathing before PDT. There were only minor variations in long-term tumor response and cure observed between the two groups tested. A second series of experiments was done to assess any treatment advantage of the adjuvant use of Fluosol-DA (20%) and carbogen breathing with PDT at high tumor photosensitizer levels. At an injected dose of 50 mg/kg of dihematoporphyrin ethers, no such advantage was observed. The administration of Fluosol-DA (20%) and carbogen breathing did not reduce the extent of PDT-induced microvascular damage, maintain high levels of tumor oxygenation through light treatment, or modify the extent of tumor cell kill following treatment.

Increases in brain tumor and cerebral blood flow by blood-perfluorochemical emulsion (Fluosol-DA) exchange.

It has been suggested that oxygen-carrying blood substitutes, perfluorochemical (PFC) emulsions, can increase blood flow and oxygen delivery to poorly perfused tumor regions. Local cerebral blood flow was measured in male Wistar rats bearing intracranial Walker 256 tumor with and without blood-PFC exchange using [14C]iodoantipyrine (IAP) and quantitative autoradiographic techniques. The exchange transfusion was performed in two groups of awake animals breathing 100% oxygen: (a) complete blood-PFC exchange, hematocrit 4%; and (b) partial blood-PFC exchange, hematocrit 20-25%. The tissue/blood partition coefficient for IAP was determined in a separate set of experiments under identical conditions and was used in calculating blood flow. Cerebral blood flow increased approximately 2-fold following complete blood-PFC exchange and 1.5-fold by the partial exchange. A similar 1.5-fold increase in flow was measured in intraparenchymal tumors following partial exchange; however, a flow increase was not identified in the meningeal extension of the tumors. The increase in cerebral blood flow is consistent with an autoregulatory response of the central nervous system vasculature to maintain an adequate supply of oxygen to central nervous system tissue. Presumably, the increase in blood flow to the intracerebral tumor reflects the autoregulatory response of the host tissue. The effect of blood-PFC exchange on blood flow and drug delivery to tumor may depend on the particular tumor and its site of growth (host tissue). The tissue/blood partition coefficient for IAP increased from 0.8 to 1.0 and 1.4 following partial and complete blood-PFC exchange, respectively. This change in the partition coefficient reflects the change in the intravascular fraction of IAP that is bound to plasma proteins. The enhanced therapeutic effect that has been reported in some experimental tumor models may result from a higher tissue/blood equilibrium distribution ratio (due to reduced plasma protein binding) resulting in a higher tissue exposure to certain drugs following PFC administration.

Effects of various oxygenation conditions on the enhancement by Fluosol-DA of melphalan antitumor activity.

The cytotoxicity of melphalan toward exponentially growing FSaIIC fibrosarcoma cells under hypoxia, normal aeration, hyperoxygenation, and stationary phase normally oxygenated cells was examined. Through 4 logs of cell kill by melphalan, there was no difference in survival of FSaIIC cells under any of the four conditions. In the fifth and sixth logs of cell kill, melphalan was most cytotoxic toward normally aerated cells. DNA alkaline elution was performed in FSaIIC cells treated for 1 h with melphalan under the various atmospheres. Both upon immediate elution and after a 6-h delay period the greatest number of DNA cross-links were formed in the normally oxygenated cells. Tumor growth delay studies of the FSaIIC fibrosarcoma treated with melphalan were performed under four levels of oxygenation. From air breathing to 100% oxygen at 3 atm, the tumor growth delay produced by melphalan increased from about 3 days to about 9 days. With the addition of Fluosol-DA, there was an increase in tumor growth delay by melphalan from about 6.5 days with air breathing to about 13 days with 100% oxygen at 3 atm (1 h). When FSaIIC fibrosarcoma tumors were treated with melphalan, and tumor cell survival was measured by colony formation in culture, increasing doses of melphalan produced increasing levels of tumor cell kill in a relatively log linear manner. The addition of Fluosol-DA to treatment with melphalan produced approximately 1 log greater tumor cell kill than melphalan and air breathing under the various oxygenation conditions. There was approximately a 4-fold increase in toxicity to bone marrow granulocyte-macrophage colony-forming units under both extended carbogen breathing conditions (6 h) and hyperbaric oxygenation conditions (100% oxygen, 1 h, 3 atm). The response of the spleen to these various treatment regimens appeared to be immediate and shortlived. Necrotic cells were seen on day 1 posttreatment, with a substantial reduction by day 4 posttreatment. Mitotic figures were essentially absent from the liver on day 1 posttreatment, but by day 4 were significantly increased in treatment groups receiving Fluosol-DA, with the largest number seen in the melphalan/Fluosol-DA with carbogen-breathing group. In conclusion, Fluosol-DA and 1 h of carbogen breathing significantly increases the antitumor activity of melphalan without a concomitant increase in normal tissue toxicity. Although increasing the oxygenation level increased the response of the tumor, normal tissue toxicity was also increased.

Effect of hypnorm, chloralosane and pentobarbital on the ultrastructure of the inner membrane of rat heart mitochondria.

Rat heart mitochondria were isolated from four groups of animals treated in a different way. The animals of the first group were killed after decapitation (D-group) without previous anaesthesia. The three other groups of animals were anaesthetised with different anaesthetics. The second group (N-group) was anaesthetised with nembutal (sodium pentobarbital), the third group with chloralosane (C-group) and the fourth group with hypnorm (H-group). From these three anaesthetics only nembutal is known to interact with mitochondria. After retrograde perfusion and excision of the heart, mitochondria were prepared from the ventricles by standard methods. After freeze-fracturing the mitochondrial suspension, the intramembrane particle dimension and density on both fracture faces of the inner mitochondrial membrane were measured. The intramembrane particle diameter on the P-face of the inner membrane of the N-group mitochondria was significantly different from D-, C- and H-group mitochondria. Also the density and diameter of the intramembrane particles on the mitochondrial inner membrane of D-group mitochondria compared to C- and H-group mitochondria were significantly different at the 95% level of confidence. Between C- and H-group mitochondria no differences of these parameters were observed. From these results it is clear that, depending on the pretreatment of the animals, a different substructure of the inner membrane of heart mitochondria is obtained.

A special lipid mixture for membrane fluidization.

The potency for membrane fluidization of mixtures containing neutral lipids (NL), phosphatidylcholine (PC) and phosphatidylethanolamine (PE) from hen egg yolk was tested on human erythrocytes and lymphocytes. A specific mixture consisting of 70% NL, 20% PC and 10% PE was found to be a potent membrane fluidizer operating almost exclusively by extracting membrane cholesterol. Spectral results and electron micrographs indicate that aqueous dispersion of this mixture consists of chylomicron-like assemblies where the neutral lipids provide the hydrophobic core on the surface of which phospholipids are spread as a monolayer.

Cardiac effects of carbon dioxide-consuming and carbon dioxide-generating buffers during cardiopulmonary resuscitation.

Recent studies have demonstrated an increase in carbon dioxide (CO2) tension (PCO2) in both mixed venous and coronary vein blood early in the course of cardiac arrest and cardiopulmonary resuscitation. Because increased PCO2 in the myocardium correlates with both ischemic injury and depression of contractile function, the effects of hypertonic solutions of either the CO2-"generating" sodium bicarbonate (NaHCO3) buffer, a mixture of sodium carbonate (Na2CO3) and sodium bicarbonate (carbicarb) acting as a CO2-"consuming" buffer, or saline placebo (NaCl) were compared during cardiopulmonary resuscitation in 25 healthy minipigs. Both buffer agents significantly increased the pH and HCO3- of arterial, mixed venous and coronary vein blood. Bicarbonate increased whereas carbicarb reduced blood PCO2 in the systemic circuit as anticipated. However, neither the PCO2 nor the lactate content of coronary vein blood was favorably altered by buffer therapy. Four of eight animals treated with bicarbonate, five of eight treated with carbicarb and six of nine placebo-treated animals were successfully resuscitated and had a comparable 24 h survival rate. Coronary perfusion pressure during precordial compression, a critical determinant of resuscitability, was transiently decreased by each of the hypertonic solutions. Accordingly, neither CO2-generating nor CO2-consuming buffers mitigated increases in coronary vein PCO2 or improved the outcome of cardiopulmonary resuscitation under these experimental conditions.

Changes in QTc interval induced with Renografin-76 and Hypaque-76 during coronary arteriography.

Renografin-76 and Hypaque-76 are both recommended for coronary arteriography. Both have the same osmolality and iodine concentration, but differ in their calcium binding properties. After selective right or left coronary arteriography in patients, Renografin-76 caused significantly more prolongation of the QTc interval than did Hypaque-76. Less calcium binding in the Hypaque formulation is probably responsible for its lesser effect on the QTc interval. This study suggests, but does not prove, that Hypaque-76 is safer than Renografin-76 for coronary arteriography.

Echocardiographic contrast agents: effect of microbubbles and carrier solutions on left ventricular contractility.

Recently, there has been a resurgence of interest in the use of contrast-enhanced echocardiography as a means of noninvasively assessing myocardial perfusion. However, if injections of echocardiographic contrast agents are to be used for this purpose it is essential that they are not intrinsically toxic to the heart. In this study, the left ventricular end-systolic wall stress-rate-corrected velocity of fiber shortening relation, a load independent index of contractility, was studied in nine dogs. Two-dimensional and targeted M-mode echocardiographic as well as central aortic pressure tracings were made during echocardiographically gated, pressure- and volume-controlled aortic root injections of nonsonicated and sonicated Renografin-76, saline and dextrose 70% (n = 6), and sonicated and hand-agitated Renografin-76/saline mixture (n = 5). Two of nine dogs received all agents. Off-line computer videodensitometric analysis documented myocardial perfusion. In all cases, data were obtained at control and 5 and 15 seconds after injection. Additional data were collected at 25 seconds after injection for the Renografin-76/saline mixture. Alterations in contractility were measured relative to control as changes in rate-corrected velocity of fiber shortening after afterload (measured as end-systolic wall stress) was eliminated as a confounding variable. Under no condition did saline or Renografin-76 cause alterations in left ventricular contractility. Nonsonicated and sonicated dextrose 70% increased left ventricular contractility at 15 seconds but not at 5 seconds after injection. Hand-agitated Renografin-76/saline mixture induced a negative inotropic effect at 5 and 15 seconds after injection.(ABSTRACT TRUNCATED AT 250 WORDS)

The search for the ideal thrombolytic agent.

Since streptokinase and urokinase became available for clinical use, numerous attempts have been made to improve these useful thrombolytic agents. To decrease its antigenicity, streptokinase has been fragmented or coupled to human plasminogen or polyethylene glycols. With a plasmin B chain-streptokinase complex a more potent agent was obtained. To prolong their half-life, streptokinase and urokinase were immobilized with water-soluble carriers. Coupling urokinase with fibrin-specific antibodies increases its thrombolytic efficacy, at least in vitro. The only thrombolytic agents with a relative fibrin specificity available for clinical purposes are tissue-type plasminogen activator and single chain urokinase-type plasminogen activator. Mutants and hybrids of these molecules are being constructed and may further improve their fibrin specificity and therapeutic potential.

Effect of exchange transfusion with Fluosol DA 20% on splenic distribution and immunocompetence of rat lymphocytes.

T- and B-splenic lymphocyte frequency, immune response 4 days after immunization with sheep red blood cells (SRBC), and proliferative response to concanavalin A (Con A) were determined 1, 3, and 5 days after exchange transfusion with Fluosol DA 20% (FDA) in adult, male Sprague-Dawley rats vs sham transfused rats. T-cell, T-helper, T-suppressor, and B-lymphocyte were reduced 1 day after transfusion (P less than or equal to 0.001). T- and B-lymphocyte frequencies were still reduced at day 3 (P = 0.0372). By day 5, there were no significant reductions in T-cell, T-helper, T-suppressor, and B-cell frequencies in the FDA-transfused rats. The frequency of cells with cytoplasmic IgG was reduced (P less than or equal to 0.025) in cells harvested from spleens of FDA-transfused rats and tested fresh. Proliferative response of splenic lymphocytes to Con A was unaffected by transfusion with FDA (P greater than or equal to 0.078). Splenic hemolytic plaques in response to SRBC were unaffected if rats were transfused 3 days after immunization with SRBC and 1 day prior to study (P = 0.941), enhanced if rats were transfused 1 day after SRBC immunization and 3 days prior to study (P = 0.0015), and suppressed if rats were transfused 1 day before SRBC immunization and 5 days before study (P less than 0.0001). Transfusion with FDA causes transient decreases in identifiable T and B lymphocytes, depresses cytoplasmic IgG-positive B cells, does not affect proliferative response to Con A, does not affect an ongoing specific immune response, enhances an early specific immune response, and inhibits the induction of a specific immune response.

Cefoxitin resistance in community-acquired gram-negative bacillary bacteremia. Associated clinical risk factors.

Among 185 patients with nonneutropenic, community-acquired gram-negative bacillary bacteremias, clinical risk factors for cefoxitin resistance included any antibiotic taken within the last three weeks (25.6% cefoxitin resistance), long-term bladder catheterization or surgical urinary diversion (23.3%), hospitalization within the last 30 days (22.9%), and nursing home residence before admission (20.8%). Patients with none of these risk factors were less likely to have cefoxitin-resistant bacteremias (0.9%). When these risk factors were examined in the subgroups of urinary tract and non-urinary tract sources of community-acquired gram-negative bacillary bacteremia, they were also helpful in predicting sensitivity to trimethoprim-sulfamethoxazole and gentamicin. The presence of one or more of the risk factors identified may be a useful adjunct in determining initial empiric antimicrobial therapy for community-acquired gram-negative bacillary bacteremia.

Induction of morphologic differentiation of endothelial cells in culture.

Human endothelial cells when grown in cell culture assume a "cobblestone" morphology and do not form tubes or capillarylike structures. We have recently identified a culture substrate containing basement membrane-derived proteins that promotes morphologic differentiation of human umbilical vein and human dermal microvascular endothelial cells into capillarylike tubes. This differentiation is rapid, beginning within 1 h and is complete by 8-12 h. On electron microscopy these cells form a lumen, derived from remodeling of multiple cells and also by forming holes in the cytoplasm of individual cells. The endothelial cells no longer proliferate when cultured on this substrate known as matrigel, but can be induced to do so when cultured on fibronectin. We have also identified a critical molecular signal for endothelial cell differentiation induced by matrigel. Laminin, a prime constituent of matrigel, and to lesser extent collagen IV appear to be key elements in the differentiation of endothelial cells induced by matrigel.

AL721, a novel membrane fluidizer.

AL721, which is a novel lipid mixture extracted from egg yolks, is believed to be a therapeutic pharmacologic agent. AL721 interacts with membranes of various types of cells with a common mode of action. AL721 modifies cellular membrane composition and fluidity through passive extraction and/or exchange of cholesterol. Physiologically diminished cell function due to rigidification of its membrane is reversible both in vitro and in vivo by AL721. Fluidization of aged membranes with AL721 has been shown to restore brain serotonin receptor function both in vitro and in vivo. AL721 can also successfully restore deficient immune responsiveness of lymphocytes to mitogen stimulation in aged subjects. Drug tolerance to morphine and ethanol develops upon elevation of the viscosity of neuronal cell membranes in order to counteract the fluidization effect of the drug. Treatment of rigidified cellular membranes with AL721 in vivo can markedly reduce withdrawal symptoms. The virucidal effect of AL721 on the human immunodeficiency virus is believed to operate by lowering of viral membrane cholesterol thus interfering with the binding of the viral antigen to the host cell. Non-toxicity of AL721 is clearly demonstrated in animal and human safety studies.

Loss of nitrogen from organs in rats induced by exogenous glucagon.

Rats weighing 220 g were injected sc with zinc protamin glucagon 20 micrograms once daily (recurrent hyperglucagonemia) and zinc protamin glucagon 60 micrograms three times daily (chronic hyperglucagonemia); the controls received the vehicle three times daily. In the first group blood glucagon rose to above 200 ng/liter for 5 h every day; in the second group it constantly stayed above 600 ng/liter. After both 2 (n = 5) and 14 (n = 5) days treatment the control total blood alpha-amino-nitrogen (AAN) concentration was 4.3 +/- 0.1 mmol/liter, and the urea nitrogen synthesis rate was 4.9 +/- 0.4 mumol/(min.100 g BW) (mean +/- SEM) in controls. In recurrent hyperglucagonemic rats, treated for both 2 (n = 5) and 14 (n = 5) days, total AAN was 3.6 +/- 0.2 mmol/liter (P less than 0.05 vs. control) and urea nitrogen synthesis rate 4.5 +/- 0.8 mumol/(min.100 g BW). In chronic hyperglucagonemic, treated for both 2 (n = 5) and 14 (n = 5) days, total AAN was 2.2 +/- 0.1 mmol/liter (P less than 0.05 vs. control) and UNSR 7.9 +/- 0.8 mumol/(min.100g BW) (P less than 0.05 vs. control). The urea excretion was identical in controls and during recurrent hyperglucagonemia, but it was increased by 50% during chronic hyperglucagonemia. Food intake was the same in all groups. N Balances decreased from 10 mmol/24 h to 5 mmol/24 h (P less than 0.05) by chronic hyperglucagonemia. The total organ N content did not change by recurrent hyperglucagonemia, but in chronic hyperglucagonemia it decreased to 65-85% (P less than 0.01) in carcass, intestines, liver, and kidneys. In conclusion chronic but not recurrent hyperglucagonemia increases the rate of urea synthesis and decreases the blood amino acid concentration. This is suggested to be a reason for the loss of N from organs by chronic hyperglucagonemia.

Highly polarized secretion of inhibin by Sertoli cells in vitro.

We have studied the regulation of inhibin secretion by rat Sertoli cells grown on extracellular matrix-impregnated porous filters in a twin chamber assembly. Previous studies have established that rat Sertoli cells cultured under these conditions reproduce the morphological and functional polarization observed in the Sertoli cell in situ. Sertoli cells isolated from 18- to 22-day-old Wistar rats were cultured for up to 8 days with daily changes of fully defined supplemented Eagle's Minimum Essential Medium (MEM). Rat inhibin was measured by RIA and pituitary cell bioassay, and transferrin by RIA. Inhibin measured by immunoassay or bioassay was always readily detectable in the upper, but not the lower, chamber. Inhibin secretion into the upper chamber exhibited a dose-dependent stimulation of up to 3.7-fold by ovine FSH, with a medium effective dose of 2.2 micrograms/liter and a constant bio- to immunoreactive ratio (3.6 +/- 0.4). Apically directed secretion accounted for over 80% of inhibit output under basal conditions and over 94% with FSH stimulation. Insulin also stimulated upper chamber inhibin secretion at a high dose (5 mg/liter) but not at lower doses or in conjunction with FSH exposure of Sertoli cells. Testosterone augmented FSH-induced stimulation of inhibin secretion, but was ineffective without FSH exposure. In contrast to inhibin secretion, for which FSH is the principal regulator, transferrin secretion by Sertoli cells is more evenly bidirectional (overall mean upper to lower chamber ratio of 1.5) and requires exposure to other stimuli (insulin, retinoic acid, and testosterone) in addition to FSH to achieve maximal secretion. Both submaximal and maximal FSH stimulation of inhibin output were augmented by a phosphodiesterase inhibitor, isobutylmethylxanthine, and these effects were fully reproduced by forskolin, which suggests the involvement of cAMP in the vectorial secretion of inhibin. The marked polarization of Sertoli cell inhibin secretion in vitro could not be explained by restricted transmembrane passage of inhibin. It is, therefore, suggested that the bulk of inhibin secretion by the immature rat Sertoli cell in vivo may be directed primarily into the seminiferous tubular lumen. Thus, in addition to its role in endocrine negative feedback signaling to the pituitary, inhibin may also have important functions in seminiferous tubular function and the support of spermatogenesis.

Differential sensitivity to beta-cell secretagogues in cultured rat pancreatic islets exposed to human interleukin-1 beta.

The early stages of insulin-dependent diabetes mellitus are characterized by a selective inability to secrete insulin in response to glucose, coupled to a better response to nonnutrient secretagogues. The deficient glucose response may be a result of the autoimmune process directed toward the beta-cells. Interleukin-1 (IL-1) has been suggested to be one possible mediator of immunological damage of the beta-cells. In the present study we characterized the sensitivity of beta-cells to different secretagogues after human recombinant IL-1 beta (rIL-1 beta) exposure. Furthermore, experiments were performed to clarify the biochemical mechanisms behind the defective insulin response observed in these islets. Rat pancreatic islets were isolated and kept in tissue culture (medium RPMI-1640 plus 10% calf serum) for 5 days. The islets were subsequently exposed to 60 pM human recombinant IL-1 beta during 48 h in the same culture conditions as above and examined immediately after IL-1 exposure. The rIL-1 beta-treated islets showed a marked reduction of glucose-stimulated insulin release. Stimulation with arginine plus different glucose concentrations, and leucine plus glutamine partially counteracted the rIL-1 beta-induced reduction of insulin release. The activities of the glycolytic enzymes hexokinase, glucokinase, and glyceraldehyde 3-phosphate dehydrogenase, were similar in control and IL-1-exposed islets. Treatment with IL-1 also did not impair the activities of NADH+- and NADPH+-dependent glutamate dehydrogenase, glutamate-aspartate transaminase, glutamate-alanine transaminase, citrate synthase, and NAD+-linked isocitrate dehydrogenase. The oxidation of D-[6-14C]glucose and L-[U-14C]leucine were decreased by 50% in IL-1-treated islets. Furthermore, there was a significant decrease in the ratios of [2-14C]pyruvate oxidation/[1-14C]pyruvate decarboxylation and L-[U-14C]leucine oxidation/L-[1-14C]leucine decarboxylation, indicating that IL-1 decreases the proportion of generated acetyl-coenzyme-A residues undergoing oxidation. However, in the presence of IL-1 there was a significant increase in L-[U-14C]glutamate oxidation. These combined observations suggest that exposure to IL-1 induces a preferential decrease in glucose-mediated insulin release and mitochondrial glucose metabolism. This mitochondrial dysfunction seems to reflect an impairment in proximal steps of the Krebs cycle. It is conceivable that the IL-1-induced suppression and shift in islet metabolism can be an explanation for the beta-cell insensitivity to glucose observed in the early phases of human and experimental insulin-dependent diabetes mellitus.