Emanuel Escher

Expression of the Five Somatostatin Receptor (SSTR1-5) Subtypes in Rat Pituitary Somatotrophes: Quantitative Analysis by Double-Label Immunofluorescence Confocal Microscopy

Using quantitative double-label fluorescence immunocytochemistry and confocal microscopy, we have analysed the pattern of expression of SSTR1-5 in normal rat pituitary somatotrophes. Antipeptide rabbit polyclonal antibodies were produced against the extracellular domains of SSTR1-5. SSTR antigens were colocalized in GH positive cells using rhodamine conjugated secondary antibody for SSTRs and FITC-conjugated secondary antibody for GH. SSTR5 was the predominant subtype which was expressed in 86 ± 9.7% of GH cells followed by SSTR2 in 42± 6.4% of GH positive cells. SSTR4 and SSTR3 were modestly expressed in 23 ± 4.7% and 18 ± 3.2% of somatotrophes respectively whereas SSTR1 was the least expressed subtype occurring in only 5± 1.2% of somatotrophes. These results demonstrate variable expression of the 5 SSTRs in somatotrophes. The preponderance of the SST-28 preferring SSTR5 subtype correlates with the reported higher potency of SST-28 than SST-14 for inhibiting GH secretion.

Visualization of neurokinin-3 receptor sites in rat brain using the highly selective ligand [3H]senktide.

The autoradiographic distribution of the neurokinin (NK)-3 receptor sub-type was visualized in the rat brain using [3H]senktide, a highly selective ligand, [3H]Senktide apparently binds to a single class of high affinity (Kd = 2.8 +/- 1.0 nM), low capacity (Bmax = 31.2 +/- 3.0 fmol/mg protein) sites in rat brain cortex. The ligand selectivity pattern reveals that eledoisin and senktide are potent competitors of both [3H]senktide and [125I]Bolton-Hunter eledoisin binding sites demonstrating the NK-3 nature of these sites. Autoradiographic data show that [3H]senktide binding sites are concentrated in mid-cortical layers, supraoptic nucleus, zona incerta, basolateral nucleus of the amygdala and interpeduncular nucleus. Much lower densities of binding are seen in most other areas such as the caudate-putamen and cerebellum. This distribution is similar, but not identical, to that previously reported for NK-3 sites using less selective ligands. It is most likely because less selective probes also bind to other classes of NK receptors. The higher selectivity of [3H]senktide is thus an important advantage for the precise characterization of NK-3 receptor binding parameters.

Substance P – Structure-Activity Studies and the Development of Antagonists

Various attempts to developing antagonists for substance P (SP) and related peptides, based on our past experience with angiotensin, kinins, and neurotensin, were unsuccessful. The particular features of SP, namely the high activity of C-terminal partial sequences in some pharmacological tests were used to develop one hexa and several octapeptide antagonists. Weak antagonists were obtained with a single modification, namely the replacement of Leu10 with trp in the sequences SP (6-11), SP (4-11) and SP (1-11). The affinity of octa and undecapeptide antagonists could be increased by using two (in positions 7 and 9 or 7 and 10) or 3 (in position 7, 9 and 10) substitutions of the natural residues with trp. Affinity of antagonists was further increased by replacing Met11 with either Nle or Phe. These new compounds showed some selectivity, [pro4, trp7 ,9, Nle11 ]-SP (4-11) being more potent in the rabbit mesenteric vein than all other octapeptides described in the present study; on the other hand, [pro4, trp7 ,9,10,Phe11]-SP (4-11) was found to be the most potent antagonist of SP and related peptides in the guinea pig ileum and the guinea pig trachea. Both compounds were similarly active in the dog carotid artery. Undecapeptide antagonists, bearing the same structural modifications as the octapeptides, were found to be stimulant in the guinea pig trachea and relaxant in the dog carotid artery. The agonistic property was eliminated by repeated applications of the compounds in guinea pig tracheae, and therefore the compounds could be tested as antagonists. The undecapeptides were found to be much more active antagonists against kasinin and eledoisin than against SP and physalaemin. The data obtained with the octa and undecapeptide antagonists of SP have been used for identification and characterization of SP receptors in various smooth muscles. It appears that SP and related peptides may exert their numerous pharmacological effects by activating more than one receptor type.

Biosensing based on surface plasmon resonance and living cells

We propose the combination of surface plasmon resonance (SPR) with living cells as a biosensing method. Our detection scheme is based on the premise that cellular activity induced by external agents is often associated with changes in cellular morphology, which in turn should lead to a variation of the effective refractive index at the interface between the cell membrane and the metal layer. We monitored surface plasmon resonance signals originating from a gold surface coated with cells on a custom apparatus after injection of various agents known to influence cellular activity and morphology. Specifically, we evaluated three types of stimulation: response to an endotoxin (lipopolysaccharides), a chemical toxin (sodium azide) and a physiological agonist (thrombin). A comparison with phase contrast microscopy reveals that SPR signal variations are associated with the induction of cell death for lipopolysaccharides treatment and a contraction of the cell body for sodium azide. Thrombin-induced cellular response shows a rapid decrease of the measured laser reflectance over 5min followed by a return to the original value. For this treatment, phase contrast micrographs relate the first phase of the SPR variation to cell contraction and increase of the intercellular gaps, whereas the recovery phase can be associated with a spreading of the cell on the sensing surface. Hence, the SPR signal is very consistent with the cellular response normally observed for these treatments. This confirms the validity of the biosensing method, which could be applied to a large variety of cellular responses involving shape remodeling induced by external agents.

Evidence for the existence of three classes of neurokinin receptors in brain. Differential ontogeny of neurokinin-1, neurokinin-2 and neurokinin-3 binding sites in rat cerebral cortex ☆

The autoradiographic distribution of the 3 neurokinin (NK) receptor sub-types, NK-1, NK-2 and NK-3, was compared in rat cerebral cortex during post-natal development using [125I]Bolton-Hunter-substance P, (2-[125I]iodohistidyl1)neurokinin A and [125I]Bolton-Hunter-eledoisin as respective radioligands. Throughout brain development, NK-1 receptor sites are present in low densities with some enrichment seen in lamina III while NK-3 binding sites are concentrated in layers IV and V. However, it appears that NK-2 receptors are mostly expressed in lamina VI and only during the first two postnatal weeks. These results demonstrate further the existence and differential ontogeny of 3 classes of NK receptors in rat brain cortex.

Receptors for substance P.I. The pharmacological preparations

The undecapeptide substance P (SP) is both a potent stimulant of intestinal (guinea pig ileum), vascular venous (rabbit mesenteric vein) and tracheal (guinea pig trachea) smooth muscles, and a relaxant of large arteries (dog common carotid artery, rabbit aorta). SP also decreases the peripheral vascular resistance in the coronary vessels of rabbit isolated hearts. The contractions of the guinea pig ileum in response to SP are partially antagonised by atropine and significantly depressed by indomethacin, while the contractions of the guinea pig trachea are strongly potentiated by indomethacin. Large arteries, but not the veins, are insensitive to SP when the endothelium is removed, and slightly less responsive to the peptide when this is applied in the presence of mepacrine, eicosatetraynoid acid or BW 755C. These results indicate that all myotropic actions of SP, except that on venous smooth muscle, are partially or completely indirect and most of them are due to the release of endogenous agents: these are acetylcholine, prostaglandins and possibly histamine in the guinea pig ileum, prostaglandins and possibly a leukotriene in the guinea pig trachea and a still unidentified factor of endothelial origin in dog and rabbit large arteries. The effect of substance P on the rabbit isolated anterior mesenteric vein appears to be a direct one.

Differential β-arrestin-dependent conformational signaling and cellular responses revealed by angiotensin analogs.

Distinct conformational changes induced in β-arrestin determine whether angiotensin II analogs enhance β-arrestin–dependent proliferation or migration. Biasing β-Arrestin Responses Activation of the angiotensin type 1 receptor (AT1R) by different ligands can trigger distinct signaling pathways and cellular responses. One such pathway is mediated by the adaptor protein β-arrestin, which can promote cellular proliferation and migration in response to AT1R activation. Zimmerman et al. found that the ability of angiotensin II analogs to enhance β-arrestin–dependent proliferation or migration was linked to distinct conformational changes in β-arrestin induced by the analogs. Thus, biased signaling downstream of the AT1R exists at the level of β-arrestin. These results may help in developing drugs that target specific signaling events triggered by this receptor without affecting others, thereby causing fewer side effects. The angiotensin type 1 receptor (AT1R) and its octapeptide ligand, angiotensin II (AngII), engage multiple downstream signaling pathways, including those mediated by heterotrimeric guanosine triphosphate–binding proteins (G proteins) and those mediated by β-arrestin. Here, we examined AT1R-mediated Gαq and β-arrestin signaling with multiple AngII analogs bearing substitutions at position 8, which is critical for binding to the AT1R and its activation of G proteins. Using assays that discriminated between ligand-promoted recruitment of β-arrestin to the AT1R and its resulting conformational rearrangement, we extend the concept of biased signaling to include the analog’s propensity to differentially promote conformational changes in β-arrestin, two responses that were differentially affected by distinct G protein–coupled receptor kinases. The efficacy of AngII analogs in activating extracellular signal–regulated kinases 1 and 2 correlated with the stability of the complexes between β-arrestin and AT1R in endosomes, rather than with the extent of β-arrestin recruitment to the receptor. In vascular smooth muscle cells, the ligand-induced conformational changes in β-arrestin correlated with whether the ligand promoted β-arrestin–dependent migration or proliferation. Our data indicate that biased signaling not only occurs between G protein– and β-arrestin–mediated pathways but also occurred at the level of the AT1R and β-arrestin, such that different AngII analogs selectively engaged distinct β-arrestin conformations, which led to specific signaling events and cell responses.

Receptors for substance P. III. Classification by competitive antagonists

A series of ten octa- and five undecapeptide antagonists of SP have been tested in four isolated smooth muscle preparations in order to characterise the receptors mediating the SP-induced contractions of the guinea pig ileum (G.P.I.), the guinea pig trachea (G.P.T.) and the rabbit mesenteric vein (R.M.V.) and the relaxation of the dog carotid artery (D.C.A.). It has been shown that: (a) Indirect effects by substance P and related peptides, mediated by acetylcholine or prostaglandins reduce the affinity of [pro4,trp7,9,10]SP (4-11) (an SP-antagonist) only in the G.P.I. (b) Octapeptide antagonists are specific for SP, have no agonistic activities and exert a competitive antagonism against SP, its homologues and fragments. (c) Undecapeptide antagonists are weaker than the octapeptides in the G.P.I. and G.P.T. and slightly stronger in the D.C.A. and R.M.V. However these compounds still have variable degrees of agonistic activity in some tissues. Affinity of octapeptide antagonists bearing the basic structure [pro4,trp7,9]SP-(4-11) is increased by the additional replacement of Leu10 with trp, Met11 with Leu, Nle or Phe, but it is slightly reduced by substituting Phe8 with Val. Antagonists containing aliphatic residues at the C-terminal end, for instance [pro4,trp7,9,Nle11]SP-(4-11) are more potent than others in the R.M.V., while those with aromatic residues, for instance [pro4,trp7,9,10]SP-(4-11) are weak on the R.M.V. but fairly active on the G.P.T. These antagonists do not show any selectivity on the G.P.I. and the D.C.A. Comparison of antagonists affinities for receptor characterisation suggest the existence of three different functional sites for SP-related peptides. The site of the G.P.I. and D.C.A., which accepts both the antagonists containing aromatic or aliphatic groups at the C-terminal end; the site of the G.P.T. which prefers the aromatic and that of the R.M.V. which shows high affinity for aliphatic residues. The receptor classification emerging from data obtained with antagonists is compared with the classifications of the literature and with those based on the order of potencies of SP homologues and fragments.

Tachykinin receptors in smooth muscles: A study with agonists (substance P, neurokinin A) and antagonists

Four preparations, sensitive to tachykinins, the guinea-pig urinary bladder, the rat duodenum, the hamster and dog urinary bladders have been investigated and compared with four other preparations described before: the guinea-pig ileum and trachea, the dog carotid artery and the rabbit mesenteric vein. On the basis of the order of potency of agonists, evaluated with substance P, physalaemin, eledoisin, kassinin and neurokinin A, the preparations can be separated into three groups, the guinea-pig urinary bladder and the dog carotid artery, in which substance P is the most potent and neurokinin A the weakest tachykinin, the rabbit mesenteric vein, the guinea-pig trachea and the rat duodenum, in which the opposite order is observed and the hamster and dog urinary bladders, in which kassinin is the most potent agonist. The guinea-pig ileum shows similar sensitivity to the five tachykinins. C-terminal partial sequences appear to be weaker than SP-(1-11) in three of the four new preparations, SP-(6-11) being first in the rat duodenum and slightly weaker than SP-(1-11) in the hamster and dog urinary bladders. Studies performed with antagonists or inhibitors of endogenous agents suggest that substance P and neurokinin A act directly on specific receptors. The effects of the two peptides are reduced by antagonists analogues of the sequence SP-(4-11). One of the antagonists, [D-Pro4,Lys6,D-Trp7,9,10, Phe11]SP-(4-11) has been shown to be competitive against substance P and neurokinin A in the guinea-pig ileum, the guinea-pig urinary bladder and the rat duodenum. This compound, shows definitely higher activity against neurokinin A and kassinin, compared to substance P in various preparations. [D-Tyr4,D-Trp7,9,Nle11]SP-(4-11) is the most potent tachykinin antagonist in the hamster and dog urinary bladders. In these preparations, the antagonists act also against substance P, but with lower affinity. These findings with antagonists support the indication, emerged from the order of potency of agonists, that tachykinins may act on two and possibly three different receptor types.

Substance P antagonists active in vitro and in vivo

[Pro4, Trp7,9]SP-(4-11) and a newly developed compound, [Pro4, Trp7,9,10]SP-(4-11) were shown to be potent antagonists of substance P (SP). They inhibited the hypotensive effect of SP in the anesthetised rats, the vasodilator effect in the rabbit perfused heart and the relaxation induced by SP in dog carotid arteries contracted with noradrenaline. Stimulatory actions of SP in the guinea pig ileum and the guinea pig trachea were also inhibited by the two antagonists, which were shown to be specific for substance P and related peptides in all the pharmacological preparations utilised in the present study.