Norbert Mayer

Effects of antibodies against nerve growth factor on the postnatal development of substance P-containing sensory neurons

Administration of anti-nerve growth factor (NGF)-antibodies to newborn rats produces a marked but reversible reduction of the substance P content in dorsal root ganglia. This is in contrast to the effect of anti-NGF-antibodies on sympathetic ganglia, where they cause a destruction of the adrenergic neurons as is evident in the irreversible reduction of tyrosine hydroxylase activity.

Characterization of porcine coronary muscarinic receptors

SummaryTo determine the muscarinic receptor subtype involved in the contractile response of coronary smooth muscle, we investigated the profiles of various muscarinic receptor antagonists competing for [3H]N-methyl-scopolamine ([3H]NMS) binding to membrane preparations from porcine coronary arteries. [3H]NMS binds to a single population of muscarinic binding sites with a KD of 135 pM and a Bmax of 57 fmol/mg. The affinity profiles of AF-DX 116 [11-2((−((diethylamino)methyl)-1-piperidinyl)acetyl)-5,11-dihydro-6H-pyrido(2,3-b)(1,4)-benzodiazepin-6-one], atropine, 4-DAMP [4-diphenylacetoxy-N-methylpiperidine methiodide], methoctramine [N,N′-bis (6-((2-methoxybenzyl) amino)hexyl)-1,8-octane-diamine tetrahydrochloride], HHSiD [hexahydrosiladi-fenidol] and pirenzepine are consistent with binding to a mixed population of muscarinic binding sites, namely of the M2 and M3 subtype.Binding curves for AF-DX 116 and methoctramine are shallow with Hill-coefficients significantly less than unity. Comparison of data from binding studies with results obtained in functional experiments, i.e. antagonism of methacholine induced contraction of porcine coronary artery rings, it was found that only the low-affinity pKi values of AF-DX 116 (6.26) and methoctramine (6.51) correlated well with functional pA2 values.It is concluded that a mixed population of the M2 and M3 muscarinic receptor subtypes is present in porcine coronary arteries. Functional experiments do not support the contribution of the M2 subtype to the contractile response. Cholinergic induced contractions of porcine coronary arteries appear to be evoked via stimulation of the muscarinic M3 receptor subtype. However, since the compounds investigated here do not markedly discriminate between cloned m3, m4 and m5 receptors the involvement of muscarinic receptors different from M1, M2 and M3 cannot be excluded.

Substance P: characteristics of binding to synaptic vesicles of rat brain.

Summary1.The binding of substance P (SP) to synaptic vesicles from rat brain was studied by use of the 125I-Tyr8-analogue of SP.2.The pH dependence of the binding of both peptides to the lipid extractable fraction of synaptic vesicles was shown to be comparable.3.The binding of 125I-Tyr8-SP shows a rate constant of association (k1=6.6×106 M−1 s−1), a rate constant of dissociation (k−1=6.4×10−4 s−1) and gives a KDof 1×10−10 M. KDderived from equilibrium studies was 3.2×10−10 M.4.The binding of 125I-Tyr8-SP to lipids of synaptic vesicles was shown to be reversible, saturable and highly specific.5.The kinetic data suggest one population of binding sites with a maximal number of 0.8 pmol per mg protein of the synaptic vesicle preparation.6.Unlabeled SP and the (2–11)-, (3–11)- and (4–11)-analogues of SP inhibit the binding of 125I-Tyr8-SP in a decreasing order in a competitive way when added in excess. Tyr8-SP and eledoisin did not interfere with the binding of 125I-Tyr8-SP whereas uperolein and neurotensin caused a partial inhibition. Physalaemin and d-Ala2-d-Met5-enkephalin enhance the binding of 125I-Tyr8-SP in a cooperative way.

Substance P in rat brain synaptosomes.

Summary1.Rat brain synaptosomes were incubated under different conditions to study the release of substance P (SP).2.Potassium ions and electrical field stimulation induced a loss of SP from synaptosomes. The release of SP by potassium in high concentrations (23.8 mM) was shown to be calcium dependent.3.Substance P was retained in synaptosomes during incubation in 0.32 M sucrose at +4° C up to 120 min. During incubation at 30° C the SP content fell initially (30 min) but was gradually restored (120 min).4.If these pre-incubated synaptosomes were reincubated for 45 min at 30° C in potassium free Krebs-Ringer-phosphate buffer a further rise in their SP content occurred which was taken as indication that SP is being synthesized in synaptosomes.5.The newly synthesized SP is presumably stored by binding to phosphatidyl serine until a sudden release is initiated by depolarization.

Cardioselectivity of AQ-RA 741, a novel tricyclic antimuscarinic drug

The interaction of the AF-DX 116 analogue, AQ-RA 741 (11-[[4-[4-(diethylamino)butyl]-1-piperidinyl]acetyl]-5,11- dihydro-6H-pyrido[2,3-b] [1,4]benzodiazepin-6-one), with muscarinic receptors, in vitro and in vivo, was examined. In radioligand binding studies, AQ-RA 741 showed high affinity for cardiac M2 sites (pKi = 8.30), intermediate affinity for cortical M1 sites (pKi = 7.70) and low affinity for glandular M3 sites (pKi = 6.82). Functional studies showed AQ-RA 741 to be a competitive antagonist and to have a 60 to 87-fold higher affinity for cardiac muscarinic receptors than for muscarinic receptors in intestinal, tracheal or bladder smooth muscle. In vivo experiments confirmed the M2 selectivity of AQ-RA 741. In rats, cats and guinea-pigs AQ-RA 741 preferentially inhibited the vagally or agonist-induced bradycardia (-log ID50 = 7.24-7.53 i.v.). The ratio of potencies observed between effects mediated by cardiac and other muscarinic receptor ranged between 9- and more than 100-fold. The results show that AQ-RA 741 is a potent and selective M2 antagonist with remarkable in vivo selectivity.

Substance P: Model studies of its binding to phospholipids

Summary1.The partition of substance P (SP) between buffer solutions (pH 1.6–7.8) and an organic, phospholipid (phosphatidyl serine, phosphatidyl ethanolamine, phosphatidyl inositol and phosphatidyl choline) containing phase (chloroform:methanol 2:1) was studied.2.The binding of SP to phosphatidyl serine, phosphatidyl ethanolamine and phosphatidyl inositol was lowest at pH 2 and increased with pH. The binding to phosphatidyl choline was much smaller and less dependent on pH.3.In contrast to the basic peptide SP (pI 10.5), physalaemin (pI 7.0) did not show any binding to phospholipids at any investigated pH value which underlines the importance of a basic group in the peptide for its binding.4.The high affinity (KD=0.1 μM) and capacity of 44 pmol SP/μg phosphatidyl serine and 48 pmol SP/μg phosphatidyl ethanolamine at pH 7.2 under conditions of saturation contrasted with the very low binding of SP to phosphatidyl inositol or phosphatidyl choline. Ionic bindings between the basic peptide and phosphatidyl serine or phosphatidyl ethanolamine are regarded to be predominant, although other binding forces cannot be excluded.5.There was a concentration-dependent reduction in the binding of SP to phosphatidyl serine or phosphatidyl ethanolamine by Na+ and Ca2+, whereas K+ showed hardly any effect at physiological concentrations.6.The model studies served to consider the possibilities of the binding of a basic peptide to lipid storage or receptor sites.

Distribution of Spinal Cord Nerve Endings Containing Various Neurotransmitters on a Continuous Density Gradient

Abstract: Homogenates of rat dorsal or ventral spinal cord were subjected to centrifugation on a continuous density gradient. The gradient was generated according to a new method with the aid of a microprocessor‐controlled HPLC pump. The distribution of substance P‐like immunoreactivity (SPI) and somatostatin‐like immuno‐reactivity (SRIFI) across the gradient showed two peaks. The SPI peak seen at lower density was found only in dorsal spinal cord tissue. No peak of SPI was seen at this position in homogenates prepared from the spinal cords of capsaicin‐pretreated rats. The second peak of SPI, found at a higher density, was accompanied by peaks in the levels of endogenous 5‐hydroxytryptamine (5‐HT), [14C]glycine, and [3H]norepinephrine uptake. This peak was seen at the same density in the dorsal and the ventral spinal cord. Tissue derived from capsaicin‐pretreated rats exhibited one peak of SPI, accompanied by a maximum of [14C]glycine uptake. The uptake of [3H]γ‐aminobutyric acid ([3H]GABA) was found to have a maximum at a somewhat lower density than that of [14C]glycine. It is concluded that the peak of SPI found at lower density in the dorsal spinal cord is associated with nerve endings belonging to capsaicin‐sensitive primary afferents, while other endings, including those also containing 5‐HT, are probably associated with the peak of SPI found at higher density.

Regional distribution and biochemical properties of 125I-Tyr8-substance P binding sites in synaptic vesicles.

Summary1.Binding of 125I-Tyr8-substance P (SP) to synaptic vesicles shows an uneven distribution within the brain and the spinal cord. The regional distribution has a positive correlation with the SP-content, except in the hypothalamus.2.Ca2+ and Mg2+-ions (1 and 10 mM) decrease the number of binding sites without alteration of affinity. EDTA and EGTA enhance SP-binding which is interpreted as being due to removal of the inhibitory influence of endogenous Ca2+ and Mg2+ through chelation with these agents. No significant inhibition of SP binding was observed by Na+ or K+ in concentrations below 100 mM.3.Pretreatment of synaptic vesicles with trypsin or with phospholipase A2, C and D leads to a total loss of SP binding showing a proteolipid or a joint protein-phospholipid nature of these binding sites. SH groups do not contribute to SP binding since no effect of N-ethylmaleimide and monoiodoacetic acid on SP binding was found.

Effect of Capsaicin Pretreatment on Substance P Binding to Synaptic Vesicles

Newborn or adult rats were pretreated with 50 mg kg–1 capsaicin. At the age of 2 to 4 months, binding of 1251‐labelled Tyr8‐substance P to synaptic vesicles prepared from different regions of the nervous system was examined. In both groups, capsaicin pretreatment led to a significant decrease in the number of binding sites in dorsal roots and spinal cord without having an effect on affinity. This decrease parallels the depletion of the substance P content (Gamse et al., 1980) and can be explained by degeneration of primary sensory neurons in newborn treated rats and by depletion of vesicles in adult treated rats.

Substance P: Binding to lipids in the brain

Summary1.Substance P (SP) could be extracted from brain homogenates with chloroform-methanol by a method which extracts all lipids.2.SP could be transferred from this total lipid extract (TLE) into an aqueous solution at low pH values (2.0–3.0).3.At higher pH values (5.5) SP could be transferred from an aqueous phase into an organic phase (chloroform: methanol, 2:1) and recombined with TLE (which was previously freed from endogenous SP) contained in this phase. The binding capacity of TLE for SP exceeded by far the amount of endogenous SP bound originally in the brain extracts.4.Among the lipids present in TLE, phosphatidylserine was able to bind and release SP in a pH dependent manner.5.It is suggested that SP bound to phosphatidylserine is the storage form of SP in the brain. The mechanisms by which it is released are still unknown. The possibility that the SP-receptor is also a phospholipid is considered.