Effect of remote ischemic conditioning on the immune-inflammatory profile in patients with traumatic hemorrhagic shock in a randomized controlled trial.

Resuscitation induced ischemia/reperfusion predisposes trauma patients to systemic inflammation and organ dysfunction. We investigated the effect of remote ischemic conditioning (RIC), a treatment shown to prevent ischemia/reperfusion injury in experimental models of hemorrhagic shock/resuscitation, on the systemic immune-inflammatory profile in trauma patients in a randomized trial. We conducted a prospective, single-centre, double-blind, randomized, controlled trial involving trauma patients sustaining blunt or penetrating trauma in hemorrhagic shock admitted to a Level 1 trauma centre. Patients were randomized to receive RIC (four cycles of 5-min pressure cuff inflation at 250 mmHg and deflation on the thigh) or a Sham intervention. The primary outcomes were neutrophil oxidative burst activity, cellular adhesion molecule expression, and plasma levels of myeloperoxidase, cytokines and chemokines in peripheral blood samples, drawn at admission (pre-intervention), 1 h, 3 h, and 24 h post-admission. Secondary outcomes included ventilator, ICU and hospital free days, incidence of nosocomial infections, 24 h and 28 day mortality. 50 eligible patients were randomized; of which 21 in the Sham group and 18 in the RIC group were included in the full analysis. No treatment effect was observed between Sham and RIC groups for neutrophil oxidative burst activity, adhesion molecule expression, and plasma levels of myeloperoxidase and cytokines. RIC prevented significant increases in Th2 chemokines TARC/CCL17 (P < 0.01) and MDC/CCL22 (P < 0.05) at 24 h post-intervention in comparison to the Sham group. Secondary clinical outcomes were not different between groups. No adverse events in relation to the RIC intervention were observed. Administration of RIC was safe and did not adversely affect clinical outcomes. While trauma itself modified several immunoregulatory markers, RIC failed to alter expression of the majority of markers. However, RIC may influence Th2 chemokine expression in the post resuscitation period. Further investigation into the immunomodulatory effects of RIC in traumatic injuries and their impact on clinical outcomes is warranted.ClinicalTrials.gov number: NCT02071290.

Human munc13 is a diacylglycerol receptor that induces apoptosis and may contribute to renal cell injury in hyperglycemia.

We have previously shown that human munc13 (hmunc13) is up-regulated by hyperglycemia under in vitro conditions in human mesangial cell cultures. The purpose of the present study was to determine the cellular function of hmunc13. To do this, we have investigated the subcellular localization of hmunc13 in a transiently transfected renal cell line, opossum kidney cells. We have found that hmunc13 is a cytoplasmic protein and is translocated to the Golgi apparatus after phorbol ester stimulation. In addition, cells transfected with hmunc13 demonstrate apoptosis after treatment with phorbol ester, but cells transfected with an hmunc13 deletion mutant in which the diacylglycerol (C1) binding domain is absent exhibit no change in intracellular distribution and no induction of apoptosis in the presence of phorbol ester stimulation. We conclude that both the diacylglycerol-induced translocation and the apoptosis represent functional activity of hmunc13. We have also demonstrated that munc13-1 and munc13-2 are localized mainly to cortical epithelial cells in rat kidney and both are overexpressed under conditions of hyperglycemia in a streptozotocin-treated diabetic rat model. Taken together, our data suggest that hmunc13 serves as a diacylglycerol-activated, PKC-independent signaling pathway capable of inducing apoptosis and that this pathway may contribute to the renal cell complications of hyperglycemia.

Dynasore enhances the formation of mitochondrial antiviral signalling aggregates and endocytosis-independent NF-κB activation.

BACKGROUND AND PURPOSE: Dynasore has been used extensively as an inhibitor of clathrin-mediated endocytosis. While studying the role of endocytosis in LPS-induced signalling events, we discovered that dynasore itself induced activation of NF-κB, independently of its effects on endocytosis and without involving the Toll-like receptor 4 signalling pathways. The purpose of this study was to characterize this novel effect and to explore the underlying mechanism of action. EXPERIMENTAL APPROACH: We utilized gel electrophoresis, microscopy, gene knockdown and luciferase-based promoter activity to evaluate the effect of dynasore on cell signalling pathways and to delineate the mechanisms involved in its effects, KEY RESULTS: Dynasore activated the NF-κB and IFN-ß pathways by activating mitochondrial antiviral signalling protein (MAVS). We showed that MAVS is activated by NOX/Rac and forms high molecular weight aggregates, similar to that observed in response to viral infection. We also demonstrated that dynasore-induced activation of JNK occurs downstream of MAVS and is required for activation of NF-κB and IFN-ß. CONCLUSION AND IMPLICATIONS: These findings demonstrate a novel effect of dynasore on cell signalling. We describe a novel Rac1-, ROS- and MAVS-mediated signalling cascade through which dynasore dramatically activates NF-κB, mimicking the viral induction of this key inflammatory signalling pathway. Our results call attention to the need for a broader interpretation of results when dynasore is used in its traditional fashion as an inhibitor of clathrin-mediated endocytosis. These results suggest the intriguing possibility that dynasore or one of its analogues might be of value as an antiviral therapeutic strategy or vaccine adjuvant.

Influences of follicle-stimulating hormone, proteases, and antiproteases on permeability of the barrier generated by Sertoli cells in a two-chambered assembly.

Factors have been identified that influence the integrity of the barrier generated by Sertoli cells (SC) in culture in a two-chambered assembly. The permeability of the barrier was assessed by determining rates of equilibration of [3H]methoxyinulin or [86Rb]Cl across the Sertoli cell monolayer. The complete system consisted of a confluent monolayer of SC maintained on an extracellular matrix (Matrigel)-coated filter together with peritubular cells on the opposite side of the filter. In confirmation of previous results, levels of plasminogen activator (PA) activity secreted were increased by treatment of SC with FSH or with cAMP derivatives [(Bu)2cAMP (dbcAMP)]. PA levels in the culture medium were inversely related to times required for 50% equilibration of [3H]methoxyinulin across the SC monolayer. Thus, elevated PA levels, elicited by stimulation with FSH or dbcAMP, were associated with a decreased integrity of the barrier generated by SC preparations maintained in serum-free medium in the complete system. The increase in permeability of the barrier in SC elicited by FSH dbcAMP could be prevented, however, by the addition of various antiproteases. FSH actions on barrier function were complex. Effects of FSH that favored barrier integrity were most readily detected when proteolytic activity was inhibited. The addition of intact serum increased the integrity of the barrier, but acid-treated serum depleted of antiproteases had no such effect. We advance the hypothesis that proteases are implicated in modulation of the formation and maintenance of the seminiferous tubule barrier by SC.

Control of levels of plasminogen activator activity secreted by Sertoli cells maintained in a two-chamber assembly.

Sertoli cells (SC), plated onto an extracellular matrix-coated membrane mounted in a two-chambered assembly, secrete both transferrin and plasminogen activator (PA) into each chamber. Although transferrin concentrations are greatest in the inner chamber, concentrations of PA activities in the outer chamber are equal to or higher than those in the inner chamber. These data indicate that transferrin and PA are preferentially secreted in different directions. The addition of FSH or cAMP derivatives stimulates the formation and secretion of tissue-type PA. Addition of FSH enhances the polarized secretion of PA into the outer chamber, as measured by elevated ratios of outer to inner compartment PA concentrations. Ratios of PA to transferrin concentrations in the outer compartment are also increased in FSH-treated preparations, demonstrating that the differential secretion of the two products is enhanced by FSH. We interpret these data to indicate that polarized SC preferentially secrete transferrin apically while preferentially secreting PA basally, and that FSH augments this polarity of SC maintained in the two-chamber assembly. The addition of peritubular cells to the system results in decreased levels of total PA activity, with greatest diminution evident in the outer compartment. Data are consistent with previous observations that peritubular cells decrease PA activity by secreting a specific inhibitor of PA. Measurements of relative amounts of transferrin and PA secreted into inner and outer chambers, respectively, provide a means to evaluate the tightness of the seminiferous tubule barrier in the model system and the extent of polarized secretion by SC in the two-chambered assembly.

Androgens inhibit plasminogen activator activity secreted by Sertoli cells in culture in a two-chambered assembly.

The addition of androgens (testosterone or dihydrotestosterone) resulted in decreased levels of detectable plasminogen activator activity in the medium when Sertoli cells were maintained in culture in a serum-free chemically defined medium in a two-chambered assembly. This occurred in the presence or absence of extracellular matrix or peritubular cells in the system. In the complete two-chambered assembly, addition of androgens simultaneously resulted in a small but significant increase in the integrity of the Sertoli cell barrier separating the two chambers, as indicated by slower rates of equilibration of [3H]methoxyinulin between inner and outer chambers. These responses to steroids appeared to be androgen specific, since other steroids examined (17 beta-estradiol, progesterone, and dexamethasone) had no detectable effects on levels of plasminogen activator activities or on barrier function. We confirmed that when FSH or (Bu)2cAMP is added to stimulate plasminogen activator secretion by Sertoli cells, the integrity of the barrier is decreased, provided no antiproteases are present in the serum-free medium. Simultaneous addition of androgens inhibited these effects of (Bu)2cAMP on Sertoli cells, but did not influence the responses of Sertoli cells to FSH. We compare actions of androgens on Sertoli cells in culture under various conditions and discuss the possible physiological significance of the inhibition of plasminogen activator activity.

Modulation of Sertoli cell functions in the two-chamber assembly by peritubular cells and extracellular matrix.

A two-chamber assembly has been employed to investigate influences of peritubular cells (PC) and extracellular matrix (Matrigel) on barrier formation by Sertoli cells (SC) in culture and on transferrin production. The kinetics of transport of [3H]inulin across a Millipore filter were essentially the same in the presence or absence of Matrigel or PC. In contrast, a SC monolayer retarded the diffusion of [3H]inulin, increasing the estimated time for 50% equilibration from about 4 h to approximately 12 h. Matrigel and PC each independently further increased the equilibration time, with the largest effects elicited by the presence of Matrigel (approximately 21 h). Data have been interpreted to indicate that these two components, especially extracellular matrix, facilitate the formation of a functional barrier by SC in the two-chamber system. PC assume a more important role than Matrigel in the modulation of transferrin secretion by SC. Transferrin concentrations were higher in the inner chamber, corresponding to those in the adluminal compartment, but transferrin masses were higher in the outer chamber under the conditions described. We report the effects of the presence and absence of Matrigel, PC, and FSH on levels of transferrin secreted by SC. Addition of FSH resulted in increased transferrin secretion by SC maintained under all conditions examined. We compare our data with those previously reported by others and attempt to provide a basis for the differences observed. We discuss the properties of the system and outline major advantages and limits of the two-chamber assembly in investigations on the polarity and properties of SC in culture.

Studies of melatonin effects on epithelia using the human embryonic kidney-293 (HEK-293) cell line.

The expression of melatonin receptors (MR) of the Mel1a subtype in basolateral membrane of guinea pig kidney proximal tubule suggests that melatonin plays a role in regulating epithelial functions. To investigate the cellular basis of melatonin action on epithelia, we sought to establish an appropriate in vitro culture model. Epithelial cell lines originating from kidneys of dog (MDCK), pig (LLC-PK1), opossum (OK), and human embryo (HEK-293) were each tested for the presence of MR using 2-[125I]iodomelatonin (125I-MEL) as a radioligand. The HEK-293 cell line exhibited the highest specific 125I-MEL binding. By intermediate filament characterization, the HEK-293 cells were determined to be of epithelial origin. Binding of 125I-MEL in HEK-293 cells demonstrated saturability, reversibility, and high specificity with an equilibrium dissociation constant (Kd) value of 23.8 +/- 0.5 pM and a maximum number of binding sites (Bmax) value of 1.17 +/- 0.11 fmol/mg protein (n = 5), which are comparable with the reported Kd and Bmax values in human kidney cortex. Coincubation with GTPgammaS (10 microM) and pertussis toxin (100 ng/ml) provoked a marked decrease in binding affinity (Kd was increased by a factor of 1.5-2.0), with no significant difference in Bmax. Melatonin (1 microM) decreased the forskolin (10 microM) stimulated cAMP level by 50%. HEK-293 cells do not express dopamine D1A receptor. Following transient transfection of HEK-293 cells with human dopamine D1A receptor (hD1A-R), exposure of the cells to dopamine stimulated an increase in the level of cAMP. Similarly, transient transfection of HEK-293 cells with rat glucagon-like peptide-1 (GLP-1), glucose-dependent insulinotropic peptide (GIP), and PTH type 1 receptors, each resulted in an hormone inducible increase in cAMP levels. Surprisingly, only the stimulatory effect of dopamine could be inhibited by exposure to melatonin. The inhibitory effect of melatonin on dopamine D1-induced increase in cAMP was completely inhibited by pertussis toxin (100 ng/ml, 18 h). Immunoblot and immunocytochemical studies were carried out using two polyclonal antibodies raised against the extra and cytoplasmic domains of Mel1a receptor. Immunoblot studies using antibody against the cytoplasmic domain of Mel1a receptor confirmed the presence of a peptide blockable 37 kDa band in HEK-293 cells. Indirect immunofluorescent studies with both antibodies revealed staining predominantly at the cell surface, but staining with the antibody directed against the cytoplasmic domain required prior cell permeabilization. By RT-PCR, HEK-293 cells express both Mel1a and Mel1b messenger RNAs, but the messenger RNA level for Mel1b is several orders of magnitude lower than for Mel1a. We conclude that HEK-293 cells express MR predominantly of the Mel1a subtype. Our evidence suggests that one of the ways that melatonin exerts its biological function is through modulation of cellular dopaminergic responses.

Controlled hot start and improved specificity in carrying out PCR utilizing touch-up and loop incorporated primers (TULIPS).

The PCR technique often yields nonspecific products. To overcome this problem, a simple, specific and efficient method was designed: touch-up and loop incorporated primers (TULIPS)-PCR. This approach utilizes loop primers (i.e., additional nontemplate 5' sequence that self-anneals to the 3' region and inhibits initiation of polymerization). Upon heating of the reaction, the primers melt, initiating hot start. The reaction also uses touch-up pre-cycling with gradual elevation in annealing temperatures to ensure correct pairing. The method has been validated with glyceraldehyde-3-phosphate dehydrogenase (GAPD) primers, and its general applicability is demonstrated by specific amplification of the human gelatinase A transgene from genomic DNA extracted from transgenic mice tails. The TULIPS-PCR protocol is a novel method. The self-annealing primers utilized in this method offer improved specificity and more robust synthesis compared with touch-down and manual hot start PCR. It is performed without the need to open, pause or add to the reaction mixture any nonrectant components, such as wax, antibody or nonspecific dsDNA.

The role of 17 beta-estradiol in the recovery from oviductal prolapse in layers.

Plasma 17 beta-estradiol concentrations, thecal estrogen content, and uterine prostaglandin synthetase activity were measured in healthy and prolapsed hens as well as in layers that recovered after exposure to low intensity lighting (250 or 50 lx). The effect of estradiol benzoate injections (100 ng, 3 X per week) in hens exposed to high intensity light (greater than 500 lx) was also studied. Prolapsed hens had significantly lower plasma 17 beta-estradiol concentrations (60 +/- 12 pg/ml; mean +/- SEM) than recovered (374 +/- 40 pg/ml) or healthy hens (475 +/- 45 pg/ml). Theca cells from recovered hens had a significantly higher content of 17 beta-estradiol (.7 ng/5 X 10(5) cells) than theca cells from normal or prolapsed birds (.3 ng/5 X 10(5) cells). Microsomes prepared from the uteri of prolapsed hens converted significantly less arachidonic acid to prostaglandin metabolites (4.4%) than did microsomes from healthy or recovered birds (9.0%). Treatment of prolapsed hens with estradiol benzoate resulted in 89% of the birds recovering within 3 weeks compared to a 4% recovery rate in the controls. We conclude that restoration of peripheral 17 beta-estradiol concentrations to normal levels was concomitant with recovery in prolapsed birds, and suggest that the estrogen exerts its effect by raising the level of prostaglandin synthetase activity in the uterus.

Description of a one step staggered reannealing method for directional cloning of PCR-generated DNA using sticky-end ligation without employing restriction enzymes.

A novel method is described for ligation of PCR products into vectors. It utilizes formation of sticky end sequences compatible with those generated on the plasmid. Four primers are used: two primers are designed to contain the desired sequence of the sticky end plus the sequence of the gene and the other two primers contain the same sequence as the first two primers without the additional bases. The primers are paired to create two PCR products containing the additional bases in a staggered 5' position. Following melting and reannealing, the newly formed products contain the additional sequences as sticky overhangs compatible with the sticky ends of the plasmid.

Partial purification of a chorionic gonadotropin-like protein from bovine cotyledons.

The presence of placental gonadotropin activity in early pregnancy was studied in the cow. Cotyledons from the first trimester of gestation were used to isolate a chorionic gonadotropin-like (CG-like) protein by means of ammonium sulfate precipitations (0.17-4.5 M) and gel filtration on Sephadex G-100 and DEAE-Sephadex. The CG-like activity was measured by a radioreceptor assay (RRA) specific for human chorionic gonadotropin (hCG). Luteotrophic activity was also assessed in two separate in vitro bioassays using cultures of bovine granulosa and rat Leydig cells. It was found that the 3 M ammonium sulfate precipitate of cotyledon extracts contained a CG-like activity of 179 mIU/mg protein as measured by RRA. The precipitate inhibited 125I-hCG binding in a linear fashion. A ninefold increase in specific activity was achieved by further purification on Sephadex G-100 (800 mIU/mg protein) and Sephadex-DEAE (1600 mIU/mg). In experiments with cell cultures, it was found that 20 micrograms of protein eluted from Sephadex-DEAE (equivalent to 16 mIU of hCG as determined by RRA) added to the culture medium significantly enhanced (P less than 0.05) progesterone production by the granulosa cells and testosterone production by the Leydig cells. We conclude that the bovine placenta contains a chorionic gonadotropin which may serve to maintain the corpus luteum of pregnancy.

Cellular activation of mesangial gelatinase A by cytochalasin D is accompanied by enhanced mRNA expression of both gelatinase A and its membrane-associated gelatinase A activator (MT-MMP).

Activation of gelatinase A represents a crucial regulatory step in the control of its enzymic activity. Rat kidney mesangial cells secrete predominantly latent gelatinase A that can be activated following treatment with cytochalasin D. In the present paper we provide new evidence, using reverse transcription-PCR, that treatment of rat mesangial cells with cytochalasin D enhances the steady-state level of mRNA of the membrane-type matrix metalloproteinase (MT-MMP), as well as of gelatinase A, with no change in the level of tissue inhibitor of metalloproteinases-2 (TIMP-2) mRNA. Since the TIMP-2 protein level is reduced in conditioned medium from cytochalasin D-treated cells, the results of the present study are consistent with a model in which the action of cytochalasin D is to cause extracellular gelatinase A and TIMP-2 to be sequestered at the plasma membrane, forming a heterotrimeric complex with MT-MMP. In this manner, TIMP-2 may assume a bifunctional role causing: (i) inhibition of gelatinase A in the extracellular compartment; and (ii) guiding gelatinase A to activation through a membrane association with MT-MMP.

Cloning of a novel gene in the human kidney homologous to rat munc13s: its potential role in diabetic nephropathy.

Glomerular mesangial cells (MC) are believed to play a pivotal role in development of diabetic nephropathy. We employed differential display reverse transcription polymerase chain reaction (DDRT-PCR) comparing human MC grown under 25 mM and 5.5 mM D-glucose and osmolarity control as a first step to identify possible candidate genes regulated by hyperglycemia. This strategy resulted in cloning of a novel gene in human MC, human munc13 (hmunc13), a human homologue of rat munc13s with the N-terminal segment similar to munc13-1 and the C-terminal segment more similar to munc13-2. Hmunc13 is also expressed in human kidney cortical epithelial cells. By using relative RT-PCR and Northern blot, we have confirmed that expression of hmunc13 in MC is up-regulated by high D-glucose treatment. Together with previous reports that munc13s binds to diacylglycerol (DAG) and that hyperglycemia increases DAG levels, these findings point to a potential role of hmunc13 in mediating some of the acute and chronic changes in MC produced by exposure to hyperglycemia.

Tyrosine kinase cell signaling pathways of rat mesangial cells in 3-dimensional cultures: response to fetal bovine serum and platelet-derived growth factor-BB.

Cells grown in 3-dimensional collagen gels adopt a nonproliferative, contractile phenotype which is more characteristic of cells in vivo than cells grown in 2-dimensional culture. The floating collagen gel contraction assay is a well-defined system used to study cell-extracellular matrix interactions grown in 3-dimensional culture. Although the cell biology of this system is well defined, the cell signaling associated with gel contraction has not been well characterized. In this study we demonstrate that fetal bovine (FBS) and platelet-derived growth factor (PDGF)-induced mesangial cell-collagen gel contraction is associated with increased tyrosine phosphorylation of a number of proteins including focal adhesion kinase (FAK) and the 42-kDa isoform of MAPK (ERK2). FBS-induced gel contraction is not affected by the presence of the MEK inhibitor PD098059. Low concentrations of PDGF-BB (10 ng/ml) induce gel contraction; however, at higher PDGF-BB concentrations (80 ng/ml) gel contraction is not observed. PDGF-BB-induced gel contraction as well as tyrosine phosphorylation of FAK are inhibited in the presence of the PI-3 kinase inhibitor wortmanin. Minimal autophosphorylation of the PDGF-beta receptor is observed under 3-dimensional culture conditions following PDGF-BB stimulation; however, when mesangial cells grown in 2-dimensional culture are exposed to PDGF-BB, the PDGF-beta receptor was prominently phosphorylated. We conclude that induction of collagen gel contraction by FBS and PDGF-BB is associated with tyrosine kinase phosphorylation and that these responses differ substantially from what occurs in 2-dimensional cultures in the presence of the same agonists.

The effect of testosterone on ram spermatozoa collective motility treated by antimycin A.

The effect of testosterone on collective motility of antimycin A-treated, Ficoll-400-washed ejaculated ram spermatozoa was studied. The lowest concentration of antimycin A which blocked collective motility in hexose deprived cells (0.4 X 10(9) cells/ml) was 2 X 10(-7) M. Fructose, glucose and mannose, but not galactose, sorbitol or inositol, served as energy sources for collective motility. Cortisol, 5-beta-DHT, androsterone and 5-alpha-pregnan 3-beta-ol-20-one did not affect collective motility. Testosterone inhibited collective motility in a dose-dependent fashion. However, it did not affect lactic acid accumulation. The effect of testosterone was noted both on cells suspended in buffer containing fructose and on exogenously starved cells treated with fructose after collective motility arrest. It was concluded that testosterone might exert its inhibitory effect on motility by uncoupling fructolytic energy production to the tail motility system.

ROS stimulate reorganization of mesangial cell-collagen gels by tyrosine kinase signaling.

Reactive oxygen species (ROS) initiate multiple pathological and physiological cellular responses, including tyrosine phosphorylation of proteins. In this study, we investigated the effects of ROS on cell-extracellular matrix interactions utilizing the floating three-dimensional collagen gel assay. Exposure of mesangial cells grown in three-dimensional culture to H2O2, 3-amino-1,2,4-triazole (a catalase inhibitor), or puromycin is associated with gel reorganization accompanied by tyrosine phosphorylation of multiple proteins, including focal adhesion kinase (FAK). Neutrophils cocultured with mesangial cells in three-dimensional culture also induce mesangial cell-collagen gel reorganization and initiate tyrosine phosphorylation of a similar set of proteins. Collectively, these results show that ROS of either endogenous or exogenous origin can modulate mesangial cell-extracellular matrix interactions through initiation of a phosphotyrosine kinase signaling cascade. Consequently, ROS may play a role as signaling molecules that regulate mesangial cell-extracellular matrix interactions in both physiological and pathological conditions.

Secretion of latent type IV procollagenase and active type IV collagenase by testicular cells in culture.

Testicular peritubular myoid cells, which have properties similar to those of vascular smooth-muscle cells, secrete a variety of metalloproteinases when maintained in culture in a chemically defined medium. The predominant metalloproteinases secreted were identified as latent type IV procollagenases having molecular masses of 72 kDa and 75 kDa, as detected in Western immunoblots with specific antibodies against type IV procollagenase. When peritubular cells were stimulated by dibutyryl cyclic AMP, forskolin or cholera toxin, they secreted increased amounts of type IV procollagenase. However, little if any of the active type IV collagenase, having a lower molecular mass of 66 kDa, could be detected under these conditions. Addition of low concentrations of cytochalasin D to peritubular cells in monoculture resulted in conversion of the latent type IV collagenase into its active form, assessed with antibody-specificity studies and by the appearance of the 66 kDa protein. In contrast, Sertoli cells in culture did not manifest an increased conversion of type IV procollagenase into type IV collagenase in the presence of cytochalasin D, even though cytochalasin D addition invariably resulted in a disruption of the microfilament assembly in each of these gonadal somatic cell populations. When peritubular cells were co-cultured with Sertoli cells, addition of cytochalasin D no longer resulted in formation of increased amounts of the active form of type IV collagenase. Sertoli cells and peritubular cells each secreted a tissue inhibitor of metalloproteinase type 2, detected with a specific antibody in a Western immunoblot to have a molecular mass of 21 kDa. We conclude that cytochalasin D acts on mesenchymal-type peritubular cells, but not on epithelial-type Sertoli cells, to enhance the conversion of latent type IV procollagenase into active type IV collagenase. This conversion of type IV procollagenase into type IV collagenase by peritubular cells was inhibited by factor(s) secreted by Sertoli cells. Interactions between Sertoli cells and peritubular cells are postulated to modulate net proteinase activities in discrete regions of the testis.