[Analysis of the causes of long-standing pelvic anterior sacral space infection and discussion of management techniques].

Objective: To investigate the causes and management of long-term persistent pelvic presacral space infection. Methods: Clinical data of 10 patients with persistent presacral infection admitted to the Cancer Hospital of Zhengzhou University from October 2015 to October 2020 were collected. Different surgical approaches were used to treat the presacral infection according to the patients' initial surgical procedures. Results: Among the 10 patients, there were 2 cases of presacral recurrent infection due to rectal leak after radiotherapy for cervical cancer, 3 cases of presacral recurrent infection due to rectal leak after radiotherapy for rectal cancer Dixons, and 5 cases of presacral recurrent infection of sinus tract after adjuvant radiotherapy for rectal cancer Miles. Of the 5 patients with leaky bowel, 4 had complete resection of the ruptured nonfunctional bowel and complete debridement of the presacral infection using an anterior transverse sacral incision with a large tipped omentum filling the presacral space; 1 had continuous drainage of the anal canal and complete debridement of the presacral infection using an anterior transverse sacral incision. 5 post-Miles patients all had debridement of the presacral infection using an anterior transverse sacral incision combined with an abdominal incision. The nine patients with healed presacral infection recovered from surgery in 26 to 210 days, with a median time of 55 days. Conclusions: Anterior sacral infections in patients with leaky gut are caused by residual bowel secretion of intestinal fluid into the anterior sacral space, and in post-Miles patients by residual anterior sacral foreign bodies. An anterior sacral caudal transverse arc incision combined with an abdominal incision is an effective surgical approach for complete debridement of anterior sacral recalcitrant infections.

[Reoperation and perioperative management of residual cyst wall with perineal intractable sinus after resection of presacral cyst tumors].

Objective: To investigate the reoperation and perioperative management of residual cyst wall with perineal intractable sinus after resection of presacral cyst tumors. Methods: The clinical data of 29 patients with residual cyst wall and perineal intractable sinus after resection of presacral cyst tumors in Affiliated Cancer Hospital of Zhengzhou University from January 2014 to August 2019 were reviewed, including the characteristics of the residual cyst wall with perineal intractable sinus after resection of presacral cyst tumors, surgical method, and perioperative management. Results: Twenty-nine patients with residual cyst wall and perineal intractable sinus after resection of presacral cyst tumors, including 9 cases of epidermoid cysts, 7 cases of dermoid cysts, 10 cases of mature teratomas and 3 cases of malignant cysts (including malignant transformation of caudate cyst and teratoma); The 29 patients underwent posterior approaches for cyst resection in other hospital before, of whom 1 patient underwent posterior combined with transabdominal approach. All of thes patients underwent resection of residual presacral cyst wall and perineal intractable sinus in our hospital, of whom 25 patients underwent a transperineal approach through an arc-shaped incision anterior to the apex of the coccyx, and the other 4 patients underwent transperineal arc-shaped incision combined with transabdominal approach. All of the patients were cured without serious complications occurring, postoperative pathological and the magnetic resonance imaging diagnosis showed that the residual cyst wall and perineal intractable sinus were all completely removed. Conclusion: Appropriate surgical approache and perioperative treatment for the patients with residual cyst wall and perineal intractable sinus are very important to promote the resection of residual cyst wall and the healing of perineal intractable sinus.

[Effect of adenosine triphosphate on type â collagen mineralization in hard tissue].

Objective: To observe the effect of adenosine triphosphate (ATP) phosphorylation on type Ⅰ collagen mineralization and explore the role of small molecule compound ATP in biomimetic mineralization. Methods: Fourier transform infrared spectroscopy (FT-IR) was used to analyze the phosphorylation of collagen molecules by different concentrations (0, 25, 50, 100 mmol/L) of ATP. The concentration of 50 mmol/L ATP was chosen to construct the phosphorylated collagen mineralization model. Transmission electron microscopy (TEM) was used to observed the ultrastructure of mineralized collagen and the collagen mineralization rate was further calculated by ImageJ software. The surface morphology of the collagen gel ATP group and the control group was observed by scanning electron microscopy (SEM) and the elemental analysis was performed by using an X-ray energy spectrometer. The artificial demineralized dentin samples were mineralized for 2 days and 4 days to compare the effect of ATP on dentin remineralization by SEM. Results: FT-IR analysis showed that the formation of new peaks at wavenumbers of 642, 818, and 902 cm(-1) indicated that ATP can phosphorylate type Ⅰ collagen. Through TEM and SEM observation, the mineralization degree of type Ⅰ collagen and demineralized dentin pretreated with 50 mmol/L ATP were significantly higher than that of the control group. Compared with the control group [(31.65±1.62)%], the mineralization rate of collagen in the ATP group [(100±0)%] was significantly increased after 2 days of mineralization (P<0.05). Conclusions: ATP phosphorylation can effectively promote the mineralization process of type Ⅰ collagen.

Effects of heat shock protein 90 expression on pectoralis major oxidation in broilers exposed to acute heat stress.

This study was conducted to determine the effects of heat shock protein 90 (HSP90) expression on pH, lipid peroxidation, heat shock protein 70 (HSP70), and glucocorticoid receptor (GR) expression of pectoralis major in broilers exposed to acute heat stress. In total, 90 male broilers were randomly allocated to 3 groups: control (CON), heat stress (HS), or geldanamycin treatment (GA). On d 41, the broilers in the GA group were injected intraperitoneally with GA (5 µg/kg of BW), and the broilers in the CON and HS groups were injected intraperitoneally with saline. Twenty-four hours later, the broilers in the CON group were moved to environmental chambers controlled at 22°C for 2 h, and the broilers in the HS and GA groups were moved to environmental chambers controlled at 40°C for 2 h. The pH values of the pectoralis major after 30 min and 24 h of chilling after slaughter of HS and GA broilers were significantly lower (P < 0.01) than those of the CON broilers. Heat stress caused significant increases in sera corticosterone and lactic dehydrogenase, the activity of malondialdehyde and superoxide dismutase, the expression of HSP90 and HSP70, and nuclear expression of GR protein in the pectoralis major (P < 0.05). Heat stress induced a significant decrease in GR protein expression in the cytoplasm and GR mRNA expression. Furthermore, the low expression of HSP90 significantly increased levels of lactic dehydrogenase and malondialdehyde and GR protein expression in the cytoplasm under heat stress (P < 0.01), and significantly decreased nuclear GR protein expression (P < 0.01). Heat shock protein 90 was positively correlated with corticosterone and superoxide dismutase activities (P < 0.01), and HSP90 mRNA was negatively correlated with pH after chilling for 24 h. The results demonstrated that HSP90 plays a pivotal role in protecting cells from oxidation.

The proteasome inhibitor bortezomib inhibits intimal hyperplasia of autologous vein grafting in rat model.

OBJECTIVE: Increasing evidence indicates that inflammation plays an important role in intimal hyperplasia (IH) induced by autologous vein grafts. The proteasome inhibitor bortezomib shows anti-inflammatory effects, so we used an autologous vein transplantation model to test whether bortezomib inhibits neointimal formation in transplant-induced vasculopathy. MATERIALS AND METHODS: We subjected 88 rats to autologous external jugular vein grafting surgery randomly assigned to be treated with bortezomib or vehicle. After 24 or 72 hours, rats were humanely killed and vein grafts processed for real-time RT-PCR (24 and 72 hours), ELISA (24 hours), or neutrophil chemotaxis assay (24 hours). Subsequently, rats were humanely killed at 1 and 2 weeks after grafting with samples processed for morphometric analysis. RESULTS: Bortezomib significantly inhibited IH at 2 weeks compared with untreated controls (P < .05). Expression of mRNA for vascular cell adhesion molecule-1, intercellular adhesion molecule-1, cytokine-induced neutrophil chemoattractant 2beta, monocyte chemoattractant-1, interleukin (IL)-1, IL-6, and tumor necrosis factor-alpha markedly increased in injured vessels during the first day after surgery declining over the following 3 days. Bortezomib significantly attenuated gene expression and protein levels of most inflammatory mediators (P < .05), simultaneously inhibiting neutrophil chemotactic activity of vessel homogenates. CONCLUSIONS: Bortezomib inhibited neointimal formation at least partially by attenuating the inflammatory response in transplant-induced vasculopathy. It may become a novel vasoprotective agent in the clinical field.

Syntheses and cytotoxicity evaluation of bis(indolyl)thiazole, bis(indolyl)pyrazinone and bis(indolyl)pyrazine: analogues of cytotoxic marine bis(indole) alkaloid.

2,4-Bis(3'-indolyl)thiazoles, 3,5-bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis(3'-indolyl)pyrazine were synthesized and evaluated for cytotoxic activity against diverse human cancer cell lines by the National Cancer Institute. These compounds demonstrated significant inhibitory effects in the growth of a range of cancer cell lines. 2,4-Bis(3'-indolyl)thiazole displayed selective cytotoxicity against certain leukemia cell lines with GI50 values in the low micromolar range while the substituted derivatives showed a broad spectrum of cytotoxic activity. 3,5-Bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis[3'-(N-methyl-indolyl)]pyrazine possessed strong inhibitory activity against a wide range of human tumor cell lines. The mechanism of action remained unknown. The results suggested that 2,4-bis(3'-indolyl)thiazoles, 3,5-bis(3'-indolyl)-2(1H)pyrazinone and 3,6-bis[3'-(N-methyl-indolyl)] pyrazine offer potential as lead compounds for the discovery of anticancer agents.

Desensitization of cardiac beta-adrenoceptor signaling with heart failure produced by myocardial infarction in the rat. Evidence for the role of Gi but not Gs or phosphorylating proteins.

This study examined mechanisms of beta-adrenergic (AR) desensitization in a myocardial infarction (MI) model of heart failure in the rat. Inotropic responses to isoproterenol (non-selective beta-AR agonist) and RO 363 (selective beta1-AR agonist), in left atria and left papillary muscle, were reduced by up to 65%, while chronotropic responses in right atria were unaffected. beta1- and beta2-AR density did not change after MI, suggesting that changes in beta-AR responsiveness are due to changes occurring downstream of the receptor. Inotropic and chronotropic responses to forskolin were not altered in right and left atria and left papillary muscle after MI, suggesting changes at the level of the G-proteins. Pertussis toxin treatment of animals restored inotropic responses to isoproterenol in left atria and left papillary muscle to levels seen in the sham group, indicating that inactivation of Gi-proteins improves inotropic function in MI rats, and that beta-ARs couple to Gi in cardiac failure. Expression of G-protein receptor kinase 2 (GRK2), beta-arrestin1 and the regulatory subunits of cAMPdPK (RI alpha and RII alpha), showed no change after MI. However the expression of Gi alpha2 was significantly increased in left ventricle (sham 0.888+/-0.140, MI 1. 759+/-0.352 P=0.026), right ventricle (sham 0.031+/-0.004, MI 0. 037+/-0.002 P=0.006) and atria (sham 0.107+/-0.006, MI 0.138+/-0.006 P=0.004), with no changes observed in the expression of Gs alpha. These results suggest that increases in Gi play an important role in the decreased beta-AR responsiveness in the rat model of MI.

Cathepsin G in degenerating and healthy discal tissue.

OBJECTIVES: To assess the eventual presence, tissue localization, molecular forms, amount and activity of cathepsin G in the annulus fibrosus. METHODS: Normal non-autolytic disc tissue was collected from cadavers within six hours after death. Degenerate disc samples were collected from low back pain patients undergoing anterior interbody fusion due to severe, discographically verified and painful disc degeneration, and from the posterior parts of intervertebral discs from 10 patients undergoing microscopic discoidectomy because of intervertebral herniation. Avidin-biotinperxidase complex staining of cathepsin G was quantitated by morphometry. Cellular localization was analyzed using double immunofluorescence staining of cathepsin G and CD68, proline 4-hydroxylase or von Willebrand factor. Neutral salt extracts were analyzed by using synthetic cathepsin G substrate in spectrophotometry, dot-immunoblotting and Western blotting. RESULTS: Histological and morphometric image analysis showed increased cellularity, increased numbers of cathepsin G positive cells and neovascularization in degenerated discs compared to control discs. Neutral salt extract of disc tissue, degenerated or normal, in contrast to control material from synovial capsular tissue, did not contain measurable cathepsin G activity, although immunoreactive enzyme was detected in dot-immunoblotting. Western blotting demonstrated that the discal cathepsin G had an apparent molecular weight of 27 kDa. CONCLUSION: Due to its properties and localization in normal and pathologically altered tissue, cathepsin G probably plays both a direct and an indirect role in extracellular matrix degradation in the annulus fibrosus. Extracted cationic cathepsin G was immunoreactive, but was functionally inhibited by serpins or, more likely, by polyanionic proteoglycans and saccharins derived from the connective tissue matrix of the annulus fibrosus.

Syntheses and biological activities of bis(3-indolyl)thiazoles, analogues of marine bis(indole)alkaloid nortopsentins.

The thiazole analogues of the marine bis(indole)alkaloid nortopsentins, 2,4-bis(3-indolyl)thiazoles, were synthesized using Hantzsch reaction between indole-3-thioamides and 3-(alpha-bromoacetyl)indoles as the key step, and these analogues showed potent cytotoxic activities against a variety of human cancer cell lines in vitro.

Nerve and blood vessel growth in response to grafted dermis and cultured keratinocytes.

The aim of this study was to study innervation and angiogenesis in response to grafts of dermis and cultured keratinocytes using immunohistochemical techniques. In a porcine model, fresh autologous de-epidermalized dermis and cultured autologous keratinocytes were combined using a two-stage technique, to produce keratodermal grafts. Wounds were encased within skin graft chambers that prevented the influence of the surrounding skin. As grafts contracted, a peripheral rim of granulation tissue became exposed, allowing us to compare the wound bed beneath grafts with that beneath the raw granulating surface. Grafts were studied for 6 weeks. Angiogenesis was studied using antisera to von Willebrand factor to detect endothelial cells. Nerve growth was studied using antisera to S-100, a Schwann cell marker, and to four axonal markers: protein gene product 9.5, C-flanking peptide of neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide. In kerato-dermal grafts (n = 28), organization of blood vessels and nerve growth occurred only beneath areas with epidermal cover as compared with the surrounding granulation tissue. Initially, the immunoreactivity to von Willebrand factor was high, but in areas with epidermal cover it assumed a more orderly pattern with fewer blood vessels. Innervation was first detected by S-100 immunoreactivity seen at 1 to 2 weeks, closely followed by that to protein gene product 9.5 and much later to calcitonin gene-related peptide. C-flanking peptide of neuropeptide Y and vasoactive intestinal peptide immunoreactivity were detected in the wound depth surrounding large blood vessels at 4 to 6 weeks. In control wounds that had been either grafted with de-epidermalized dermis alone (n = 10) or allowed to granulate (n = 10), persistently there was high immunoreactivity to von Willebrand factor but minimal immunoreactivity to the neural markers. In conclusion, kerato-dermal grafts become innervated, and beneath their surface there is also vascular organization to resemble normal skin. Keratinocytes themselves may influence angiogenesis and innervation, as both processes failed to occur beneath granulating areas.

Morphological changes of neural and vascular peptides in human skin suction blister injury.

Suction blister injury is an experimental model for the investigation of the possible derangement of dermal/epidermal interaction in injury. An extensive fibre network can be stained in skin using antisera to the pan-neuronal marker protein gene product 9.5 (PGP) and the sensory neuropeptide calcitonin gene-related peptide (CGRP), while microvessels are identifiable with antisera to the endothelial marker von Willebrand's factor (vWf) and the peptide endothelin (ET). To investigate the possible involvement of superficial cutaneous innervation and microcirculation during the repair process in injury, human skin biopsies taken at different times after suction blister injury were investigated by quantitative immunohistochemistry. Neural and endothelial changes were seen in both edge and blister areas. PGP- and CGRP-immunoreactive nerves showed an increase in both areas compared with control skin up to 6 h after injury, followed by a decrease which lasted until 72 h. This was followed by a gradual increase of both nerve types starting from the blister edge and lasting up to 8 days after injury when the values were similar to controls. Similarly, in the blister area of the skin, vWF-immunoreactive capillaries showed statistically significant increases between 0 and 6 h after injury, followed by a decrease at 12 and 18 h which was maintained up to 72 h. ET-1 immunoreactivity showed a similar, although more variable, pattern of changes. At the blister edge from 23 h onwards, both vWf and ET-1 immunoreactivities showed a second increase from the edge of the blister spreading towards the centre of the blister ahead of the nerve increase. This lasted up to 8 days, when vWf immunoreactivity showed a statistically significant increase compared with control skin. Generally the numerical increase observed at early time points was accompanied by a strong staining intensity, which reverted to a normal staining intensity at later time points. These results suggest that there is a reactive process for all immunoreactive elements in the early stages of injury, followed by changes indicative of neuronal and endothelial damage (depletion phase) and the subsequent repair process. The repair started with re-epidermalization from the blister edge, followed by revascularization and, lastly, by reinnervation of the tissue. These results indicate a close relationship between epidermis, blood vessels, and nerve fibres during the healing process which follows suction blister injury.

Early increase precedes a depletion of VIP and PGP-9.5 in the skin of insulin-dependent diabetics--correlation between quantitative immunohistochemistry and clinical assessment of peripheral neuropathy.

Diabetic neuropathy affects both sensory and autonomic peripheral nerve fibres. Vasoactive intestinal polypeptide (VIP) is present in autonomic fibres which modulate sweat secretion, while calcitonin gene-related peptide (CGRP) is localized to cutaneous sensory fibres. In this study, immunohistochemistry and image analysis were used to assess changes of VIP and CGRP, and of the pan-neuronal marker protein gene-product (PGP)-9.5, in skin biopsies of 18 patients affected by type 1 diabetes (age range 18-46 years) and from seven aged-matched controls. Patients were divided into three groups: group 1 (n = 6), with diabetes for 6 months to 3 years; group 2 (n = 5), with the disease for 5-10 years; and group 3 (n = 7), with diabetes for more than 10 years. VIP immunoreactivity (IR) and PGP-9.5-IR were significantly reduced around sweat glands (P < 0.005) in groups 2 and 3. Epidermal CGRP-IR and PGP-9.5-IR were significantly reduced in group 3 (P < 0.05). Twenty-eight per cent (5/18) of all patients showed high VIP-IR around sweat glands (> 95 per cent confidence limits of controls) and all of these patients had diabetes for less than 3 years. Conversely, 55 per cent (10/18) of patients had low VIP-IR (< 5 per cent confidence limit of controls). The latter, compared with the former, showed a significantly longer duration of diabetes (Fisher exact test P = 0.002), presence of clinical autonomic neuropathy (Fisher exact test P = 0.04), and a reduced sural nerve conduction velocity (Fisher exact test P = 0.04). These results suggest that quantitative immunohistochemical analysis of peptide-containing cutaneous nerves allows an objective evaluation of nerve fibre alterations at early stages of diabetes than is currently possible with neurophysiological functional tests.

Immunohistochemical measurements of nerves and neuropeptides in diabetic skin: relationship to tests of neurological function.

Image-analysis was used to measure nerves immunoreactive to the general neuronal marker protein gene product 9.5 (PGP 9.5-IR) and the neuropeptides calcitonin gene-related peptide and vasoactive intestinal polypeptide in standardised leg skin biopsies of three age-matched groups of young subjects: non-diabetic (n = 14), diabetic patients with normal small fibre function ("non-neuropathic", (n = 11) and diabetic patients with abnormal small fibre function ("neuropathic", n = 11). Depletion of nerves and neuropeptides was most marked in the epidermis, where calcitonin gene-related peptide-immunoreactivity was more frequently absent than PGP 9.5-IR in diabetic patients. Epidermal PGP 9.5-IR nerve area and counts were reduced in neuropathic compared with normal subjects (p less than 0.001), as were epidermal calcitonin gene-related peptide nerve counts (p = 0.003). Sweat gland PGP 9.5 and vasoactive intestinal polypeptide, which may be involved in sweat production, showed no diminution in diabetic patients (area: p = 0.160, p = 0.372 by ANOVA). Two diabetic patients showed elevated sweat gland PGP 9.5-IR and three had increased sweat gland vasoactive intestinal polypeptide; this may represent nerve proliferation. In local sweat tests, acetylcholine-stimulated sweat output was associated with increased immunoreactivity, while the sympathetic skin response showed inverse correlations with immunoreactivity. There were no consistent changes with other commonly-used neurophysiological tests. HbA1 correlated negatively with immunohistochemical measurements. Neuropeptide changes were seen in the absence of macro- and microvascular disease, and epidermal nerve depletion occurred in patients with normal thermal thresholds and cardiac autonomic function.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of age on endothelin-1 binding sites in rat cardiac ventricular membranes.

To establish whether the density, affinity, or selectivity of endothelin-1 (ET-1) binding sites in cardiac ventricular membranes varies with age, membranes were harvested from 5- to 7-day-, 20-day-, and 8- to 9-week-old Sprague Dawley rats and labeled with [125I]ET-1. Selectivity was established by using cold ET-1, ET-2, ET-3, big ET-1, and (+)PN200-110 to inhibit specific binding of [125I]ET-1. Over the age span studied, selectivity and affinity of the [125I]ET-1 binding sites was unchanged, but density (Bmax) decreased from 209.7 +/- 18.4 at 5-7 days to 154.0 +/- 8.9 (p less than 0.02) at 20 days, and to 89.7 +/- 5.2 (p less than 0.01) fmol/mg protein at 8-9 weeks. These age-dependent differences in Bmax were not accompanied by a change in membrane yield and occurred at a time when the specific binding of (+)[3H]PN200-110 increased.

Reoxygenation, but neither hypoxia nor intermittent ischemia, increases [125I]endothelin-1 binding to rat cardiac membranes.

Standard binding techniques were used to establish whether either hypoxia, reoxygenation, perfusion under acidotic conditions, or "stunning" of the myocardium resembles ischemia and postischemic reperfusion in increasing cardiac membrane [125I]endothelin-1 (ET-1) binding site density (Bmax). Membranes from aerobically perfused rat hearts bound [125I]ET-1 to a single population of sites, with an affinity (KD) of 0.093 +/- 0.004 nM and a Bmax of 98.8 +/- 5.2 fmol/mg of protein. Bmax was increased (p less than 0.01) after 30 min of global ischemia, and further increased upon reperfusion, without changes in KD or selectivity. Neither three 10 min episodes of ischemia separated by 15 min of perfusion, nor perfusion at pH 6.8 instead of 7.4, nor 60 min of hypoxia altered Bmax, KD, or selectivity. Reoxygenation after 60 min of hypoxia increased Bmax (p less than 0.01) and KD (p less than 0.01) without changing selectivity. These results are interpreted to mean that the ischemia-induced increase in Bmax for [125I]ET-1 cannot be explained simply in terms of the ischemia-induced acidosis, or the accompanying reduction in tissue adenosine triphosphate and creatine phosphate.

125I-endothelin-1 binding to brain and cardiac membranes from normotensive and spontaneously hypertensive rats.

125I-labelled endothelin-1 was used to identify specific high affinity endothelin-1 binding sites in left ventricular and brain membranes harvested from female age-matched normotensive Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR). Relative to results obtained for brain membranes from normotensive rats, brain membranes from hypertensive rats exhibited an increase in 125I-endothelin-1 binding site density (P less than 0.02). In left ventricular membranes from the hypertensive rats 125I-endothelin-1 binding site density was reduced (P less than 0.02), relative to the density in ventricular membranes of normotensive rats. These results indicate that hypertension may have an organ specific effect on 125I-endothelin-1 binding site density.

Cyclosporine increases endothelin-1 binding site density in cardiac cell membranes.

When used in high concentrations, the immunosuppressive agent cyclosporine A has toxic side effects, including cardiac fibrosis and calcification. Cyclosporine-treated mice were used to investigate whether a cyclosporine dose regime which produces cardiac fibrosis and calcification alters the density of the specific binding sites for the endothelial-derived vasoconstrictor polypeptide, endothelin-1. After twenty-one days of treatment the endothelin-1 binding site density in cardiac cell membranes was increased, without any change in selectivity. This increase in endothelin-1 binding site density could contribute to the cytotoxic effects of cyclosporine.

Specific high-affinity binding sites for 125I-labelled porcine endothelin in rat cardiac membranes.

125I-labelled porcine endothelin (125I-endothelin) was used to identify specific high affinity endothelin binding sites in rat cardiac membrane fragments. Binding was to a single population of sites, with a KD of 0.20 +/- 0.03 nM and a Bmax of 93.5 +/- 6.4 fmol/mg protein at 37 degrees C. Reducing the temperature to 25 degrees C increased (P less than 0.02) the KD without changing Bmax. 125I-Endothelin binding was Ca2+ independent. Specific binding was saturable and displaceable by cold endothelin and sarafotoxin S6b, but not by (-)Bay K8644, nicardipine, (-)D888, (+)cis-diltiazem, prenylamine, lidoflazine, flunarizine, nor by 10(-10)-10(-4) M CoCl2, nor 10(-10)-10(-4) M NiCl2. omega-Conotoxin, prazosin, isoprenaline, angiotensin II and its inhibitor, vasopressin and its inhibitor, glyceryl trinitrate, amiloride, ergometrine and FII stonefish toxin also failed to displace bound 125I-endothelin. 10(-4)-10(-2) M CaCl2, 10(-4)-10(-2) M MgCl2, 3 X 10(-6)-10(-3) M MnCl2, 10(-5)-3 X 10(-4) M NiCl2, and 3 X 10(-5)-3 X 10(-4) M CoCl2 stimulated the binding. Incubation at 100 degrees C for 10 min destroyed specific binding.

Sarafotoxin S6c is a relatively weak displacer of specifically bound 125I-endothelin.

Sarafotoxin S6a, S6b and S6c are chemically related vasoconstrictor polypeptides obtained from the venom of the snake, Atractaspis engaddensis. Each contains twenty one amino acid residues, two intrachain cysteine linkages and a long hydrophobic tail. Structurally these polypeptides resemble endothelin. Binding studies with 125I-endothelin showed that 125I-endothelin bound to rat ventricular membranes is totally displaceable by sarafotoxin S6b and endothelin, with IC50 values of 0.21 and 0.16 nM, respectively. Sarafotoxin S6c, which differs from sarafotoxin S6b in containing threonine instead of serine at residue 2, arginine instead of lysine at residue 4, and glutamic acid instead of lysine at residue 9, only weakly displaced bound 125I-endothelin (IC50, 854 nM). These results indicate that the ability of the sarafotoxins to interact with the endothelin binding site is not solely dependent on the long hydrophobic tail or the cysteine linkages.