The effect of pentosan polysulphate (SP54) on the fibrinolytic enzyme system. An experimental animal study

Pentosan polysulphate (SP54) causes a transient increase in blood fibrinolysis in conscious and anaesthetized rats. Postoperative "fibrinolytic shutdown" was prevented by a dose of 2 mg/kg body weight but there appeared to be no dose-response relationship with higher doses. Fibrinolysis was also measured in conscious unstressed animals using an indwelling jugular cannula. The venous response to SP54 in these animals was substantially higher than the arterial response. Experiments with an inferior vena cava model of thrombosis suggest that a single dose of 10 mg/kg pentosan polysulphate given 90 min after thrombus formation is sufficient to achieve thrombolysis. This effect was more marked if the animals were given multiple doses over 24 to 48 hours.     

The effect of pentosan polysulphate (SP54) on the human fibrinolytic system

In human volunteers the oral and subcutaneous (s.c.) administration of SP54 enhanced the fibrinolytic potential of plasma. When the effect was compared between exercise and s.c. administration of SP54 the latter was found to "peak" at about 3 hours and to have a more sustained effect than the exercise. Oral versus s.c. administration suggests that only about 10% of the SP54 reaches the circulation by the oral route. However, repeat SP54 oral dosage did not elicit any resistance to the daily response over a period of five days and suggests that the volunteers used in this study tolerated repeated use of the drug with no ill-effects. The source of the fibrinolytic enhancement is as yet not fully explained and a hypothesis is presented which suggests that very small elevations of t-PA in plasma can affect the fibrinolytic potential in the presence of forming fibrin but may remain undetected by conventional immunometric assays.     

Effects of various doses of SP 54 on fibrinolytic activity in patients with thrombotic diseases

SP 54 was synthetized more than 30 years ago, but the proper oral dosage has not been established yet. Therefore we gave two doses. 150 and 500 mg to patients suffering from various arterial and venous thrombotic diseases, for investigating the dose-response effect. 16 patients received 150 mg, 14 patients 500 mg and 10 in each group both doses. We could observe significant changes in ELT, WBL and plasminogen level, but the increase of fibrinolytic activity was not dose-dependent in most cases. It indicates that an individual dosage is needed. Therefore we suggest an "SP 54 loading test" before prescribing the drug.     

Effect of S 17092, a novel prolyl endopeptidase inhibitor,on substance P and alpha-melanocyte-stimulating hormone breakdown in the rat brain

In the present study, we have investigated the effects of a novel prolyl endopeptidase (EC 3.4.21.26, PEP) inhibitor, compound S 17092, on substance P (SP) and alpha-melanocyte-stimulating hormone (alpha-MSH) metabolism in the rat brain. In vitro experiments revealed that S 17092 inhibits in a dose-dependent manner PEP activity in rat cortical extracts (IC50 = 8.3 nm). In addition, S 17092 totally abolished the degradation of SP and alpha-MSH induced by bacterial PEP. In vivo, a significant decrease in PEP activity was observed in the medulla oblongata after a single oral administration of S 17092 at doses of 10 and 30 mg/kg (-78% and -82%, respectively) and after chronic oral treatment with S 17092 at doses of 10 and 30 mg/kg per day (-75% and -88%, respectively). Concurrently, a single administration of S 17092 (30 mg/kg) caused a significant increase in SP- and alpha-MSH-like immunoreactivity (LI) in the frontal cortex (+41% and +122%, respectively) and hypothalamus (+84% and +49%, respectively). In contrast, chronic treatment with S 17092 did not significantly modify SP- and alpha-MSH-LI in the frontal cortex and hypothalamus. Collectively, the present results show that S 17092 elevates SP and alpha-MSH concentrations in the rat brain by inhibiting PEP activity. These data suggest that the effect of S 17092 on memory impairment can be accounted for, at least in part, by inhibition of catabolism of promnesic neuropeptides such as SP and alpha-MSH.     

Bronchial responses to substance P after antigen challenge in the guinea-pig: in vivo and in vitro studies

The effect of antigen challenge on the airway responses to substance P and on the epithelial neutral endopeptidase (NEP) activity was investigated in aerosol sensitized guinea-pigs. In vivo, bronchial responses to aerosolized substance P were similar to the responses observed in antigen-challenged guinea-pigs and in the control groups. In contrast, when the guinea-pigs were pretreated with the NEP inhibitor, phosphoramidon, a significant increase in the airway responses to substance P was observed after antigen challenge in vivo. However, in vitro, the contractile responses of the tracheal smooth muscle to substance P were similar between groups of guinea-pigs, in respect to the presence or absence of the epithelium and/or phosphoramidon. Histological studies showed an accumulation of eosinophils in the tracheal submucosa after antigen challenge and intact epithelial cells. These results show that in vivo bronchial hyperresponsiveness to substance P after antigen challenge in the guinea-pig is not associated with increased responses of the smooth muscle to exogenous SP in vitro. In addition, the results with phosphoramidon suggest that loss of NEP activity cannot account for the in vivo bronchial hyperresponsiveness to substance P presently observed.     

Influence of passive avoidance learning by substance P in the basolateral amygdala

Neuropeptide substance P (SP) has reinforcing and memory facilitating effects after its peripheral or central application. Rats self-inject SP into the ventromedial caudate-putamen and SP microinjections into the basal forebrain induce place preference with a simultaneous increase of dopamine level. In the amygdaloid body SP positive neurones and terminals have been identified. The aim of the present study was to examine the possible reinforcing effects of SP in the basolateral amygdala (ABL). CFY male rats were conditioned in two-compartment passive avoidance paradigm and place preference was examined in two-compartment-box and in circular open field. Animals were microinjected bilaterally with 10 ng SP, 100 ng SP or vehicle solution (0.4 microl/side) into the ABL. Results showed that post-shock infusion of 10 ng SP significantly enhanced passive avoidance learning while 100 ng SP was ineffective. In two-compartment-box and in circular open field place preference did not develop after SP treatments, however. Our data are the first to demonstrate that SP in the ABL is involved in learning and memory processes related to aversive situations. Results that SP microinjections were not followed by rewarding-reinforcing consequences in place preference paradigms indicate that the local SP network in the ABL is not involved in neuronal circuitry responsible for addictive behaviour.     

Stimulation of epithelial cell growth by the neuropeptide substance P

The neuropeptide substance P (SP) was found to stimulate DNA synthesis and cell growth for epithelial cells (cornea and lens) in a serum-free environment. The length of treatment time was shown to be important since longer times shifted the dose-response curve to the left. In short-term DNA synthesis studies (40 h) the stimulation with SP (or synergism with insulin) was not apparent until close to 10 μM, however, when DNA synthesis assays were carried out over a long period of time (5 days) stimulation with SP was seen at 1 pM. The stimulation of DNA synthesis by SP was synergistic with insulin for lens epithelial cells, but little synergism was seen with corneal epithelial cells. It cell growth studies on lens epithelial cells SP also showed growth stimulation by itself and synergism with insulin at concentrations of 1–2 pM. The neuropeptide calcitonin gene related peptide (CGRP) showed no DNA synthesis stimulating ability on epithelial cells by itself at concentrations as high as 2.5 μM; however, it was synergistic with SP at a concentration of 0.025 μM. SP pretreatment of epithelial cells for 2 h causes an increase in cellular sensitivity to subsequent addition of either SP or insulin. This increase is consistent with the hypothesis that either the signal from SP persists after its removal from the cell or the dissociation time for SP from its receptor is longer than the wash time.     

Anti-inflammatory Effect of Tanshinone I in Neuroprotection Against Cerebral Ischemia–Reperfusion Injury in the Gerbil Hippocampus

Tanshinone I (TsI) is an important lipophilic diterpene extracted from Danshen (Radix Salvia miltiorrhizae) and has been used in Asia for the treatment of cerebrovascular diseases such as ischemic stroke. In this study, we examined the neuroprotective effect of TsI against ischemic damage and its neuroprotective mechanism in the gerbil hippocampal CA1 region (CA1) induced by 5 min of transient global cerebral ischemia. Pre-treatment with TsI protected pyramidal neurons from ischemic damage in the stratum pyramidale (SP) of the CA1 after ischemia–reperfusion. The pre-treatment with TsI increased the immunoreactivities and protein levels of anti-inflammatory cytokines [interleukin (IL)-4 and IL-13] in the TsI-treated-sham-operated-groups compared with those in the vehicle-treated-sham-operated-groups; however, the treatment did not increase the immunoreactivities and protein levels of pro-inflammatory cytokines (IL-2 and tumor necrosis factor-α). On the other hand, in the TsI-treated-ischemia-operated-groups, the immunoreactivities and protein levels of all the cytokines were maintained in the SP of the CA1 after transient cerebral ischemia. In addition, we examined that IL-4 injection into the lateral ventricle did not protect pyramidal neurons from ischemic damage. In conclusion, these findings indicate that the pre-treatment with TsI can protect against ischemia-induced neuronal death in the CA1 via the increase or maintenance of endogenous inflammatory cytokines, and exogenous IL-4 does not protect against ischemic damage.     

Preincubation with Substance P Induces Substance P-Stimulated Phosphatidylinositol Turnover in Cultured Cerebellar Astrocytes

In this study we have investigated the effects of preincubation of cultured astrocytes with substance P (SP) on subsequent 125I-Bolton-Hunter conjugated SP (125I-BHSP) receptor binding, and SP-stimulated phosphatidyl-inositol (PI) accumulation. Spinal cord astrocytes preincubated for up to 96 h with SP (0.001-1,000 nM) suffered a dose-dependent decrease in both subsequent 125I-BHSP and SP-stimulated PI turnover. In contrast, preincubation of cerebellar astrocytes with SP resulted in an increase in SP-stimulated PI turnover, with no change in 125I-BHSP receptor binding. SP-induced PI turnover in cerebellar astrocytes was maximal after 72 h of preincubation with 0.1 nM SP. These data suggest that increased coupling between receptor and second messenger occurs in response to chronic exposure to SP.     

Effect of substance P on the retention of a brightness discrimination task in rats

The effect of substance P (SP) on acquisition and retention of a footshock-motivated brightness discrimination was investigated in rats, 250 micrograms SP/kg were injected intraperitoneally either 30 min before or immediately after the training session. Acquisition of the brightness discrimination was not affected by SP administered 30 min before training. However, both the pre-training and post-training injections of SP resulted in a significant improvement of retention tested 24 h after training. The effect of SP on memory consolidation is discussed.     

Effect of the N-terminal fragment of substance P on the microcirculatory system

Biomicroscopic experiments have shown that the N-terminal fragment of substance P (SP1-4), when applied to the rat mesentery, has a considerably lower injuring effect than substance P (SP1-11) itself. SP1-4 activity, as compared to SP1-11 activity regarded as 1, was 0.007 in case of microcirculatory disturbances and venular permeability increase and 0.0007 in case of mast cell degranulation increase. The data obtained suggest that the slightest damaging effect of SP1-4 on microcirculation is combined with anti-stress activity.     

Prostaglandin involvement in lung C-fiber activation by substance P in guinea pigs.

Airway hyperresponsiveness is a cardinal feature of asthma. Lung C-fiber activation induces central and local defense reflexes that may contribute to airway hyperresponsiveness. Initial studies show that substance P (SP) activates C fibers even though it is produced and released by these same C fibers. SP may induce release of other endogenous mediators. Bradykinin (BK) is an endogenous mediator that activates C fibers. The hypothesis was tested that SP activates C fibers via BK release. Guinea pigs were anesthetized, and C-fiber activity (FA), pulmonary insufflation pressure (PIP), heart rate, and arterial blood pressure were monitored before and after intravenous injection of capsaicin (Cap), SP, and BK. Identical agonist challenges were repeated after infusion of an antagonist cocktail of des-Arg9-[Leu8]-BK (10(-3) M, B1 antagonist), and HOE-140 (10(-4) M, B2 antagonist). After antagonist administration, BK increased neither PIP nor FA. Increases in neither PIP nor FA were attenuated after Cap or SP challenge. In a second series of experiments, Cap and SP were injected before and after infusion of indomethacin (1 mg/kg iv) to determine whether either agent activates C fibers through release of arachidonic acid metabolites. Indomethacin administration decreased the effect of SP challenge on FA but not PIP. The effect of Cap on FA or PIP was not altered by indomethacin. In subsequent experiments, C fibers were activated by prostaglandin E2 and F2alpha. Therefore, exogenously applied SP stimulates an indomethacin-sensitive pathway leading to C-fiber activation.     

β2SP/TET2 complex regulates gene 5hmC modification after cerebral ischemia

βII spectrin (β2SP) is encoded by Sptbn1 and is involved in the regulation of various cell functions. β2SP contributes to the formation of the myelin sheath, which may be related to the mechanism of neuropathy caused by demyelination. As one of the main features of cerebral ischemia, demyelination plays a key role in the mechanism of cerebral ischemia injury. Here, we showed that β2SP levels were increased, and this molecule interacted with TET2 after ischemic injury. Furthermore, we found that the level of TET2 was decreased in the nucleus when β2SP was knocked out after oxygen and glucose deprivation (OGD), and the level of 5hmC was reduced in the OGD+β2SP KO group. In contrast, the expression of β2SP did not change in TET2 KO mice. In addition, the 5hmC sequencing results revealed that β2SP can affect the level of 5hmC, the differentially hydroxymethylated region (DhMR) mainly related with the Calcium signalling pathway, cGMP-PKG signalling pathway, Wnt signalling pathway and Hippo signalling pathway. In summary, our results suggest that β2SP could regulate the gene 5hmC by interacted with TET2 and will become a potential therapeutic target for ischemic stroke.     

Effect of substance P on the monoamine-containing cells of the taste buds

The effect of substance P (SP) on monoamine-containing cells of the frog taste buds was studied by fluorescent microscopy. Intraperitoneal injection of SP resulted in a gradual increase of cell serotonin content. In monoamine deficiency caused by previous injection of rausedyl, SP favoured the recovery of the serotonin level to initial. When SP and rausedyl were used combined, SP protected the serotonin-containing cells from the depleting effect of rausedyl. The functional role of SP in the taste apparatus is discussed.     

Selective disappearance of two secreted host proteins in the course of Semliki Forest virus infection of Aedes albopictus cells.

One secreted host protein of molecular weight 54,000 (SP 54) disappeared (from 24 to 48 h after infection) in Semliki Forest virus-infected Aedes albopictus cell clone C6/36 grown in both Mitsuhashi-Maramorosch basal medium and tissue culture medium 199 and reappeared when cells went into the permanently infected state. C6/36 is a high virus producer showing a cytopathic effect. A second secreted host protein of molecular weight 62,000 (SP 62) was prominent if cell clone C6/36 was grown in tissue culture medium 199. After infection in this medium, the protein showed a behavior similar to that described for SP 54. These secreted proteins were not affected in two original Aedes albopictus cell lines. SP 54 and SP 62 are monomeric proteins and structurally not related.     

Substance P Enhances Desensitization of the Nicotinic Response in Bovine Chromaffin Cells but Enhances Secretion upon Removal

A fundamental process in neurosecretion is desensitization, or a declining response to a stimulus. The response of chromaffin cells to continuous nicotinic stimulation, secretion of catecholamines, desensitizes within a few minutes. The neuropeptide substance P (SP) has been reported to prevent desensitization in culture dish experiments and to enhance desensitization in patch clamp studies. In the present study, these contradictory responses have been demonstrated and the apparent contradictions resolved. We have measured catecholamine secretion by on-line electrochemical detection in a constant-pressure flow system. Isolated chromaffin cells cultured on quartz plates were stimulated with the nicotinic agonist 1,1-dimethyl-4-phenylpiperazinium (DMPP) in the presence and absence of SP. SP inhibited secretion and increase the rate of desensitization compared with stimulation by DMPP alone. However, when the cells were stimulated a second time with DMPP alone immediately after 5-min stimulation with SP + DMPP, the rate of desensitization was markedly lower than the control. Removal of SP after a desensitizing stimulation with SP + DMPP caused a slow secondary release of catecholamine in response to the continued stimulation with DMPP. The kinetic analysis of the secretory response shows that the primary response to SP is enhanced desensitization, but that upon removal of SP the response to DMPP desensitizes less rapidly. We suggest that SP protects some receptors from nicotinic desensitization while holding them in an inactive state, and that upon removal of SP these receptors can slowly respond to DMPP.     

Enhanced ganglionic responses to substance P in spontaneously hypertensive rats

Intravenous injection of substance P (SP) increases blood pressure in normotensive rats by stimulating sympathetic ganglia. This study compared the effects of SP to increase renal nerve firing and blood pressure in normotensive and hypertensive rats treated with chlorisondamine. The increase in renal nerve firing was greatest in spontaneously hypertensive rats (SHR), intermediate in Wistar rats, and least in Wistar-Kyoto (WKY) rats. Blood pressure was increased more in SHR than in Wistar rats. Blood pressure was not increased in WKY rats. Responses to the ganglionic stimulant 1,1-dimethyl-4-phenylpiperazinium were the same in the three strains. These results suggest that there is a selective increase in the action of SP on sympathetic ganglia of SHR and that ganglion responsiveness to SP is correlated with its effect on blood pressure.     

Clinical experience with heparinoid sodium pentosan polysulfate (SP54) in the treatment of cerebral ischemia

Fifty-five patients suffering from acute cerebral ischaemia were treated with pentosan polysulphate (SP54) intravenously and subsequently orally. The control group consisting of 35 patients received the infusion solution and vasodilator drugs. The statistically significant changes appearing following infusion of SP54 include the following parameters: haematocrit, blood viscosity and cholesterol concentrations. In individual cases the euglobulin lysis time was shortened within two hours after the beginning of the infusion. As demonstrated by the statistically reliable results in patients treated with SP54 the neurological symptoms improved significantly within the first two weeks after the onset of the disease compared to the control groups. Based on case reports, recommendations are given for the indication and contraindication of SP54 treatment in cerebral ischaemia.     

Acute effect of cigarette smoke on cytoplasmic motility of alveolar macrophages in dogs

To study effects of cigarette smoke on the cytoplasmic motility (CM) of alveolar macrophages (AM), we measured remanent field strength (RFS) in dogs in vivo. Four days after instillation of ferrimagnetic particles (Fe3O4, 3 mg/kg) into the right lower lobe bronchus, RFS was measured at the body surface immediately after magnetization of the Fe3O4 particles by an externally applied magnetic field. RFS decreased with time due to particle rotation (relaxation), which is thought to be inversely related to CM of AM (J. Appl. Physiol. 55: 1196-1202, 1983). The initial relaxation curve was fitted to an exponential function. The relaxation rate (lambda 0) increased during cigarette smoke inhalation and returned to base-line values within 15 min. With the inhalation of the smoke of up to five cigarettes, peak lambda 0 was increased; with a further increase in the number of cigarettes, the effect of cigarette smoke decreased or disappeared. Nicotine injection and acetylcholine inhalation increased respiratory resistance to a degree similar to that observed with cigarette smoke but did not change lambda 0. However, either substance P (SP) or capsaicin injection increased lambda 0 in a fashion similar to that noted with cigarette smoke inhalation. Repeated administration of SP produced a significant tachyphylaxis of the effect, and capsaicin did not increase lambda 0 after the cigarette smoke-induced tachyphylaxis of the effect. Colchicine inhibited the cigarette smoke-induced increase in lambda 0. These results suggest that cigarette smoke increases CM of AM, probably through the release of tachykinins including SP from sensory nerves in the lung.