Carlo Alberto Maggi

Nitric oxide mediates angiogenesis in vivo and endothelial cell growth and migration in vitro promoted by substance P.

We evaluated the effects of nitric oxide (NO) generators and endogenous production of NO elicited by substance P (SP) in the angiogenesis process. Angiogenesis was monitored in the rabbit cornea in vivo and in vitro by measuring the growth and migration of endothelial cells isolated from coronary postcapillary venules. The angiogenesis promoted in the rabbit cornea by [Sar9]-SP-sulfone, a stable and selective agonist for the tachykinin NK1 receptor, and by prostaglandin E1 (PGE1), was potentiated by sodium nitroprusside (SNP). Conversely, the NO synthase inhibitor N omega-nitro-L-arginine methyl ester (L-NAME), given systemically, inhibited angiogenesis elicited by [Sar9]-SP-sulfone and by PGE1. Endothelial cells exposed to SNP exhibited an increase in thymidine incorporation and in total cell number. Exposure of the cells to NO generating drugs, such as SNP, isosorbide dinitrate, and glyceryl trinitrate, produced a dose-dependent increase in endothelial cell migration. Capillary endothelial cell proliferation and migration produced by SP were abolished by pretreatment with the NO synthase inhibitors N omega-mono-methyl-L-arginine (L-NMMA), N omega-nitro-L-arginine (L-NNA), and L-NAME. Exposure of the cells to SP activated the calcium-dependent NO synthase. Angiogenesis and endothelial cell growth and migration induced by basic fibroblast growth factor were not affected by NO synthase inhibitors. These data indicate that NO production induced by vasoactive agents, such as SP, functions as an autocrine regulator of the microvascular events necessary for neovascularization and mediates angiogenesis.

The mammalian tachykinin receptors

The tachykinins (TKs) are a family of small peptides which share the common C-terminal sequence Phe-X-Gly-Leu-MetNH2. Three peptides of this family, substance P, neurokinin A and neurokinin B, have an established role as neurotransmitters in mammals. 2. Three receptors for TKs have been cloned: they are G-protein coupled receptors with seven putative transmembrane spanning segments and have been termed NK1 (substance P-preferring), NK2 (neurokinin A-preferring) and NK3 (neurokinin B-preferring). 3. Synthetic agonists are available to selectively stimulate only one receptor, while natural TKs can act as full agonist at each one of the three receptors, albeit at different concentrations. 4. A number of potent and selective antagonists, both peptide and nonpeptide in nature, have recently been developed. 5. The introduction of these ligands has revealed an unforeseen pharmacological heterogeneity of NK1, NK2 and NK3 receptors which appears largely, if not exclusively, linked to the existence of species homologues of the three receptors.

The effects of tachykinins on inflammatory and immune cells

The aim of this article is to provide an up-dated overview of the available information on the role played by tachykinins in recruiting/regulating the function of immune/inflammatory cells, an issue which has received considerable input from the recent availability of potent and selective antagonists for tachykinin receptors. It appears that NK1 receptors play a role in mediating the extravascular migration of granulocytes into inflamed tissues in response to various inflammatory stimuli, although this effect may not be due to the expression of NK1 receptors by granulocytes themselves. Several data also imply a role for NK1 and NK2 receptors in regulating immune function. No data are available to suggest the expression of NK3 receptors by inflammatory/immune cells. Mast cell degranulation by substance P appears to be a non-receptor dependent response which may take place in vivo during intense stimulation. An emerging concept in the field relates to the ability of certain immune cell types to synthesize and possibly release tachykinins. Immune cells could represent an additional source of tachykinins in inflamed tissues, providing a non-neurogenic tachykininergic contribution to the local inflammatory process.

Substance P stimulates neovascularization in vivo and proliferation of cultured endothelial cells

We have investigated the possible effect of substance P (SP), a main mediator of neurogenic inflammation, on the growth of capillary vessels in vivo, and on the proliferation of cultured endothelial cells in vitro. Slow release preparations of SP were implanted into the avascular cornea of New Zealand White rabbits and vessel growth was monitored daily through a slit lamp stereomicroscope. SP (1-5 micrograms/pellet) induced a marked neovascularization. A selective NK-1 receptor agonist [beta-Ala4, Sar9, Met(O2)11]-SP(4-11) also induced neovascularization. The addition of SP to serum-free cultured endothelial cells, isolated from bovine adrenals (BACE) and from human umbilical cord veins (HUVE), increased proliferation of both cell lines in a concentration-dependent manner with maximal activity at 10(-8) M (BACE) and 10(-10) M (HUVE). The selective NK-1 receptor agonist induced a similar proliferative action on both cell lines, while the selective NK-2 receptor agonist [beta-Ala8]-NKA(4-10) and the selective NK-3 receptor agonist [MePhe7]-NKB had no significant effect. Two different SP antagonists [D-Pro2, D-Trp7,9]-SP and [D-Pro4, D-Trp7,9,Phe11]-SP (4-11) blocked the response to SP. These findings indicate that SP can directly stimulate the process of neovascularization, probably through induction of endothelial cell proliferation. This hitherto unraveled activity of SP could play a key role in the trophic action produced by activation of the efferent function of peripheral endings of primary sensory neurons.

Nitric Oxide Promotes Proliferation and Plasminogen Activator Production by Coronary Venular Endothelium Through Endogenous bFGF

We reported previously that NO is responsible for the angiogenesis produced by endothelium-dependent vasodilating peptides. To investigate the mechanisms by which NO controls angiogenesis, NO was assessed for the ability to affect cell proliferation and upregulation of urokinase-type plasminogen activator (uPA) induced by basic fibroblast growth factor (bFGF) when added exogenously to or when produced endogenously by coronary venular endothelial cells (CVECs). The treatment of the cells with the NO donor sodium nitroprusside (NaNp) induced uPA upregulation and cell proliferation, which were prevented by anti-bFGF antibodies. Similarly, the NO-dependent mitogenic activity of the vasodilating peptide substance P (SP) was blocked by anti-bFGF antibodies, thus implicating endogenous bFGF in the NO-induced response. NaNp and SP induced bFGF expression as measured by Western blot analysis of CVEC extracts and by differential reverse transcriptase-polymerase chain reaction of bFGF mRNA. SP-induced upregulation of bFGF was prevented by the NO synthase inhibitor N omega-monomethyl-L-arginine. We conclude that NO promotes cell proliferation and uPA upregulation in CVECs by inducing endogenous bFGF and that this pathway mediates the angiogenetic response to the vasoactive neuropeptide SP. This signaling paradigm may provide an important link between shear rate, NO, bFGF, and coronary angiogenesis.

Competitive antagonists discriminate between NK2 tachykinin receptor subtypes

1 We have compared the ability of various tachykinins and selective tachykinin receptor agonists to induce contraction of the endothelium‐denuded rabbit pulmonary artery (RPA) and hamster trachea (HT) and have estimated the affinity of some newly developed NK2 selective antagonists in the same tissues. 2 In confirmation of previous findings, experiments with the agonists indicated that NK2 receptors are the main if not the sole mediators of the response to tachykinins in both RPA and HT. No evidence for significant degradation of neurokinin A (NKA) was found in either tissue when experiments were repeated in the presence of a mixture of peptidase inhibitors (thiorphan, captopril and bestatin, 1 μm each). 3 The peptide antagonists tested were: Peptide I = [Tyr5, d‐Trp6,8,9, Arg10]‐NKA(4–10); Peptide II = [Tyr5, d‐Trp6,8,9, Arg10]‐NKA(3–10); Peptide III = Ac‐Leu‐Asp‐Gln‐Trp‐Phe‐Gly‐NH2. The three peptides produced a concentration‐dependent rightward shift of the concentration‐response curve to NKA in both RPA and HT with no significant depression of the maximal response attainable. The slopes of the Schild plots were not significantly different from unity, indicating a competitive antagonism. Peptides I and II were about 100 times more potent in the RPA than in the HT, while Peptide III was about 100 times more potent in the HT than RPA. 4 The pA2 values obtained in these two tissues with the three antagonists were not significantly different when tested in the absence or presence of peptidase inhibitors, or when a selective NK2 receptor agonist, [βAla8]‐NKA(4–10) was used instead of NKA. Similar pA2 values were obtained after 15 or 90 min of incubation with the antagonists. Peptides I, II and III had no inhibitory effect on contractions produced by noradrenaline in the RPA or by carbachol in the HT. 5 Peptides I, II and III showed weak or no antagonistic activity toward the vasodilatator effect of substance P in the dog carotid artery (NK1 receptor‐mediated) or toward the contractile effect of neurokinin B in the rat portal vein (NK3 receptor‐mediated). 6 These results provide pharmacological evidence for heterogeneity of NK2 receptors in the RPA and HT. The NK2 receptors present in these tissues are not discriminated by natural tachykinins or selective agonists, but are recognized with very different affinity by NK2 receptor antagonists.

The nonstop transvesical cystometrogram in urethane-anesthetized rats: A simple procedure for quantitative studies on the various phases of urinary bladder voiding cycle

A new cystometric procedure that involves a nonstop transvesical (through a needle inserted into the bladder dome) infusion of saline in urethane-anesthetized rats is described. This permits the obtainment of a series of repetitive voiding cycles in both male and female rats. Recording at high-chart speed permits quantitative measurements of the various phases of the voiding cycle. Micturition was almost invariably associated with appearance, during a sustained increase of intravesical pressure, of a series of high-frequency oscillations, which were paralleled by a stream-like emission of the infused fluid. Micturition was not observed after topical tetrodotoxin, after bilateral transection of pelvic nerves, or in acute spinal rats. Intravenous hexamethonium produced a dose-related impairment of the voiding cycle, and, at high doses, suppressed micturition, and overflow incontinence ensued. The experimental procedure described herein appears to be suitable for physiopharmacological studies dealing with regulation of voiding cycle.

Prostanoids as local modulators of reflex micturition

Surge of interest about the possible role of prostanoids in the regulation of urinary bladder function can be traced back to the early 1970s, after the demonstration that prostaglandin-like material is released in the circulation during or immediately after bladder distension. The discovery that even a mild form of tissue stimulation, such as stretch of smooth muscle cells, releases significant amounts of prostanoids prompted the speculation that locally generated prostaglandins (PGs) might aid the receptive functions of hollow organs, such as the stomach and the urinary bladder, thereby allowing accommodation of their physiological content. While the latter interpretation turned out to be incorrect for the urinary bladder, these earlier studies attracted the attention of many investigators on the possible role of prostanoids as local modulators of micturition. This topic has been the subject of some recent reviews addressing the effect of prostanoids in regulating motility, blood flow, defence against infection and carcinogenesis in the urinary bladder. The aim of this short review is to re-address the topic of prostanoid modulation of reflex micturition with particular emphasis on a target for prostanoid action in the urinary bladder (i.e. sensory nerves) which, until recently, has been somehow neglected.

Distribution of calcitonin gene-related peptide-like immunoreactivity in various rat tissues: correlation with substance P and other tachykinins and sensitivity to capsaicin

Calcitonin gene-related peptide-like immunoreactivity (CGRP-LI) has been measured in various tissues of control rats and rats pretreated with systemic capsaicin, s.c. (50 mg/kg as newborns or as adults, 125 mg/kg as adults) and compared with the tissue level of substance P- and tachykinin-like immunoreactivities (SP-LI and TK-LI). The rank order of CGRP-LI concentration in various tissues was as follows: trigeminal ganglion greater than urinary bladder greater than ureter much greater than distal duodenum much greater than proximal duodenum much greater than skin (snout) greater than thymus = right atrium = vas deferens. A complete depletion of CGRP-LI following capsaicin treatment of both adult and newborn animals was observed in urinary bladder, ureter, atrium, vas deferens and skin. Capsaicin pretreatment of both adult and newborn rats reduced CGRP-LI in the duodenum by about 50%. CGRP-LI in trigeminal ganglion was reduced only in newborn animals, while it was not affected in the thymus. The CGRP-LI/SO-LI ratio varied in these tissues between 33.2 (urinary bladder) and 0.9 (proximal duodenum). A significant correlation was found between CGRP-LI and SP-LI or TK-LI in tissues where immunoreactivities were depleted by capsaicin, as well as in the urinary bladder of individual animals. The correlation between CGRP-LI with SP-LI and TK-LI upon treatment with capsaicin indicates that neurons containing SP and TK as well as CGRP, and neurons containing CGRP only, are affected in a similar manner by capsaicin.

The effects of topical capsaicin on rat urinary bladder motility in vivo

The effects of topical capsaicin on urinary bladder motility were investigated following saline-induced distension of the bladder wall in urethane-anaesthetized rats and compared to the effects of topical substance P and acetylcholine. Capsaicin and substance P produced similar excitatory effects in both quiescent and rhythmically contracting bladders, i.e., a TTX resistant tonic contraction followed by a series of rhythmic, TTX sensitive, phasic contractions. Acetylcholine, in doses equieffective in producing TTX resistant contractions was less effective than capsaicin or substance P in triggering neurogenic rhythmic contractions of bladder muscle. Atropine pretreatment prevented the neurogenic component of the excitatory effect of both capsaicin and substance P. Repeated applications of capsaicin but not of substance P led to desensitization. Bladders of animals pretreated (4 days before) with a large dose of s.c. capsaicin developed insensitivity to topical capsaicin and a larger volume of saline was required to trigger neurogenic rhythmic contractions of the detrusor muscle. These results suggest that capsaicin acts by interfering with the mechanism(s) regulating the threshold for the micturition reflex to occur.