Beta-endorphin. Biological activity of analogs containing dermorphin and dynorphin sequences: ileum and vas deferens assays

Biological activity of synthetic beta-endorphin (beta-EP) analogs containing dermorphin or dynorphin-A-(1-13) structure has been investigated using the guinea pig ileum and the vas deferens of the mouse, rat and rabbit. Replacement of NH2-terminal 1-7 segment of camel beta-EP [beta c-EP-(1-7)] with dermorphin caused a great increase in opiate potency of the analog. [Dermorphin (1-7)]-beta c-EP was 120 times more potent than beta c-EP in the guinea pig ileum assay, 49 times more potent in the mouse vas deferens assay; and only 4 times more potent in the rat vas deferens assay. Replacement of NH2-terminal 1-13 segment of human beta-EP [beta h-EP-(1-13)] with dynorphin-A-(1-13) caused an increase in opiate potency in both the guinea pig ileum and rabbit vas deferens assays, a complete loss of potency in the rat vas deferens assay, and no change in the mouse vas deferens assay. In comparison with dynorphin-A-(1-13), the hybrid peptide was less potent in the guinea pig ileum assay as well as in mouse and rabbit vas deferens assay. It is suggested that beta c-EP-(8-31) facilitates the dermorphin moiety to act on opiate mu and delta receptors but not on the epsilon receptor, while beta h-(14-31) reduces the action of dynorphin on mu, delta and kappa receptors.     

Comparative effects of somatostatin and enkephalins on the guinea pig ileum and the rat vas deferens

The action of somatostatin (SRIF (somatotrophin release inhibiting factor)) was compared with that of Met-enkephalin (Tyr-Gly-Gly-Phe-Met) in the electrically stimulated guinea pig ileum myenteric plexus longitudinal muscle and with that of an enkephalin analogue (FK 33-824 (Tyr-D-Ala-Gly-MePhe-Met-(O)-ol)) in the rat vas deferens. In both tissues SRIF produced a twitch inhibition which was not antagonized by naloxone and which showed a long-lasting tachyphylaxis. The enkephalins tested produced a naloxone-antagonizable inhibition of twitch in both tissues but no tachyphylaxis. Therefore we conclude that SRIF is not acting at opiate receptors in these tissues.     

Calcium antagonists and contractile responses in rat vas deferens and guinea pig ileal smooth muscle.

The effect of depletion of extracellular Ca2+ (Ca2+ext) on the loss of responsiveness of the guinea pig ileal longitudinal muscle (g.p.i.l.m.) and the rat vas deferens (r.v.d.) to K+ and cis-2-methyl-4-dimethylaminomethyl-1,3-dioxolane methiodide (CD), and K+ and noradrenaline (NA), has been examined and compared with the effects of a variety of local anesthetics and calcium antagonists. The results indicate that qualitative similarities are apparent with respect to the dependence of agonist-induced activity on Ca2+ext in both the g.p.i.l.m. and r.v.d. Distinct differences, however, in the Ca2+ translocation processes in these two tissues, in response to the different agonists, can be shown by the use of a variety of 'calcium antagonists' thus indicating that such translocation processes are both tissue and agonist selective. It is thus noted that, contrary to the Ca2+ depletion studies, D 600 and the usually more potent BAY-1040 showed no discrimination of action or potency in their ability to inhibit components of the NA response in the r.v.d. In contrast, D 600 and the more potent BAY-1040 selectively inhibited the tonic component of the K+ response. Treatment with SKF 525A and parethoxycaine (PC) in the g.p.i.l.m. and SKF 525A in the r.v.d. resulted in a nonselective inhibition of responses of the tissues to all stimulants. However, in the r.v.d. PC potentiated NA action, and its methobromide (MeBr) derivative potentiated both NA and K+ action and also, like PC, partially shifted to the left the dose-response curve to Ca2+ in NA-depolarizing Ca-free Tyrode's. The quaternary MeBr and the tertiary 2-chloroethyl (2Cl) derivatives of SKF 525A and PC were selectively more effective against CD- than K+ supported contractile activity in the g.p.i.l.m. and the 2Cl derivatives were more effective against NA than K+ responses in the r.v.d. The 2Cl derivative of PC also was more effective in antagonizing the Ca2+ dose-response curve in high-CD or high-NA than in high-K+ Ca2+-free Tyrode's.     

Pharmacological reactions of the circular muscle of the guinea pig vas deferens.

Pharmacological responses of spiral strips prepared from the guinea pig vas deferens to various adrenergic and cholinergic agonists and autacoids were studied. On the circular muscle alpha adrenergic, muscarinic cholinergic and histaminergic receptors were identified. The responses evoked on the circular and longitudinal muscles were of the same type.     

The activity of synthetic leukotriene C-1 on guinea pig trachea and ileum

The effects of synthetic leukotriene C-1 (LTC-1) on isolated guinea pig trachea and ileum have been determined and compared to histamine. LTC-1 produced a slow contraction of the trachea and the ileum with pD2 values of 8.7 +/- 0.1 (n = 14) and 8.5 +/- 0.1 (n = 13), respectively. In comparison, the pD2 values for histamine were 5.6 +/- 0.1 (n = 6) and 6.2 +/- 0.1 (n = 6), indicating LTC-1 was 2-3 orders of magnitude more potent. LTC-1 was antagonised by FPL 55712 with pA2 values of 6.9 +/- 0.1 (n = 5) and 6.4 +/- 0.1 (n = 7) on the trachea and ileum, respectively. Incubation with lipoxidase produced a time and enzyme dependent loss of biological activity and a concurrent shift in U.V. absorption spectrum.     

Adrenergic presynaptic receptors: examination of a hypothesis in guinea pig vas deferens.

The hypothesis was examined that phenoxybenzamine enhances both the overflow of noradrenaline and the mechanical response in guinea pig vas deferens by blockade of presynaptic inhibitory receptors located on adrenergic nerve terminals which serve a negative-feedback function. Preparations were stimulated with a constant small number of pulses but at three different frequencies (1, 5, and 15 Hz) and the relative effectiveness of phenoxybenzamine in enhancing overflow assessed. According to the presynaptic receptor hypothesis inhibition of transmitter output should increase with increasing frequency due to increased activation of receptor sites by endogenously released noradrenaline. The antagonist enhanced the overflow of tritium but did so to a similar extent at all three frequencies, regardless of the length of the interval between pulses. Similarly, no evidence for a greater sensitization of the mechanical response by phenoxybenzamine at the higher frequencies was obtained. The conditions of the present experiment were considered optimal for the operation of the negative-feedback system and the results indicate that the physiological relevance of such a system is questionable.     

Desensitization of the alpha adrenergic receptor system in guinea pig vas deferens

Desensitization induced by alpha adrenergic (alpha-Ad) stimulation was investigated in organ cultured vas deferens of guinea pig. Brief exposure (1-2 min) of the muscle to noradrenaline (NA) caused short-term desensitization to both NA and acetylcholine (ACh), but not to high K+. After removing the agonist this desensitization completely disappeared within 15 min. Prolonged exposure to NA (i.e., cultured with NA for 3-24 hr) elicited long-term desensitization to NA, ACh and K+ (50 mM), but it did not change the maximal contraction by high K+ (154 mM). After removing NA from the culture medium the response to the agonist was restored to normal within 24 hr, but not within 15 min. The number and affinity of alpha-Ad and muscarinic ACh receptors, which were measured by the binding of 3H-WB4101 and 3H-QNB, respectively, were not changed in the muscle during these treatments. Moreover, long-term desensitization, but not short-term desensitization, was depressed by the concomitant presence of cycloheximide. The possible mechanisms of desensitization were discussed in comparison with those of various receptor systems.     

Action of colchicine on cholinergic and adrenergic mechanisms in guinea pig vas deferens

Guinea pig vas deferens responds to externally applied acetylcholine (ACh) or noradrenaline (NA) by a small rapid contraction (phasi phase) and then a large contraction (tonic phase). The phasic phase was not affected by removal of external Ca²⁺, but tonic phase depended on external Ca²⁺. At lower temperatures the two components became larger and detectable separately. The tonic phase induced by ACh at low temperature (at 20°C) was greatly depressed by brief treatment with colchicine (0.5 μM – 5 μM), although the tonic phase at high temperature (at 37°C) was not affected. Na-induced contraction (phasic or tonic phase) was not changed by the colchicine-treatment. High K⁺ (40 mM)-contracture, which in many cases consisted of a single phase and depended on external Ca²⁺, was also not affected by brief treatment with colchicine. Culture of vas deferens for 3 days in the presence of colchicine, increased the phasic phase of ACh- and NA-induced contractions significantly, but reduced the tonic phase of contractions induced by ACh and NA. Colchicine also reduced high K⁺-contracture, the decrease depending on the period of culture with colchicine. Organ culture with colchicine did not affect the amounts of m-ACh and α-Ad receptors or the IC₅₀ value of ACh and NA on ³H-ligand binding. These results suggest that colchicine specifically interacts with some steps in m-ACh and α-Ad receptor-responsor (e.g. ionophore) coupling without affecting the receptor number or affinity of the receptors for agonists. The mechanisms of action of colchicine are discussed in relation to m-ACh and α-Ad receptor functions.     

Opioid activity of delta O-endorphin in the guinea pig ileum.

The oligopeptides beta- and delta O-endorphin were isolated from porcine and bovine pituitary respectively. Their opiate activity was determined in the guinea pig ileum and compared to that of the pentapeptide methionine-enkephalin and morphine. The rank order of opioid activity was found to be: morphine greater than beta-endorphin = Met-enkephalin greater than delta O-Endorphin which lacks the four C-terminal amino acids of beta-endorphin displayed 60% of the activity of beta-endorphin. These results indicate, that C-terminal amino acids contribute little to the affinity of beta-endorphin for opiate receptors in the guinea pig ileum.     

beta-Endorphin: structure-activity relationships in the guinea pig ileum and opiate receptor binding assays.

The opiate activities of some derivatives and enzymatic digests of camel and human β-endorphin were determined in the guinea pig ileum and rat brain opiate receptor binding assays. Derivatives of β-endorphins altered within the amino-terminal five residues showed pronounced losses in activity. Anisylation of the C-terminal glutamic acid residue of β_h-endorphin produced only small reductions in activity. Chymotryptic digestion greatly weakened the opiate activities of β_h-endorphin, whereas carboxypeptidase A, tryptic and leucine aminopeptidase digests showed only small losses in potency. The C-terminus of β-endorphin appears to contribute little directly to opiate activity. Amino acid analysis and assay of the leucine aminopeptidase digests suggest that the larger potency of β-endorphin relative to Met-enkephalin may be a consequence of its greater resistance to exopeptidase attack.     

Binding and biologic activity of neurotensin in guinea pig ileum

Experiments were performed to relate receptor binding to biologic activity for the contractile effect of neurotensin (NT) in guinea pig ileum. The contractile response was examined on pieces of ileum under 1 g tension in a 5 ml bath of oxygenated Tyrode's at 38°C. NT contracted the longitudinal muscle (ED₅₀, 0.3 nM), the 2–3 g response peaking at 1 min and fading rapidly. In the presence of atropine (1 μM), ≥50% of the response was blocked and the residual effect gave an ED₅₀ of 1.4 nM. In the presence of atropine and CP-96,345, a substance P receptor antagonist (0.2 μM), no contraction was observed at 20 nM NT. Thus, there were two components to the response, one involving acetylcholine (ED₅₀, 0.3 nM) and one substance P (ED₅₀, 1.4 nM). Using membrane preparations and ¹²⁵I-labeled NT, specific, high affinty receptors for NT were demonstrated in the muscle and myenteric plexus. Scatchard analyses indicated the presence of two binding sites (K_ds: 0.1 nM and 2 nM). Sodiu ion and GTP analogs inhibited binding. Binding and biologic activity were similar in regard to dependence on specific groups within NT and sensitivity to metal ions. The high potency of Hg⁺⁺ was consistent with an involvement of free sulfhydryl group(s) in the binding reaction; this was supported by work with SH-directed agents. The results suggest that two receptor types or configurations may mediate the two components of the contractile effect of NT on guinea pig ileum.     

Beta-endorphin. Synthesis and biological activity of analogs with disulfide bridges

Two analogs of human beta-endorphin (beta-EP) which contain cystine bridges, [Cys15-Cys26,Phe27,Gly31]-beta-EP (I) and [Cys16-Cys26,Phe27,Gly31]-beta-EP (II), were synthesized by the solid-phase method. Peptides I and II were shown to contain 2-2.5 times the opiate receptor binding activity of beta-endorphin. We also synthesized two analogs with reduced alkylated cysteine residues and these peptides, [Arg9,19,24,28,29 Cys(Cam)11,26,Phe27,Gly31] and [Arg9,19,24,28,29,Cys-(Cam)12,26,Phe27,Gly31], were shown to have approximately the same opiate receptor activity as beta-endorphin.     

Carbonic anhydrase--marker of cell type in cell culture of the guinea pig vas deferens]

Polyclonal antibodies (PCAB) to smooth muscle myosin (SMM), monoclonal antibodies (MCAB) to cytokeratin 8 (clon HI, IgGI) and H4 (IgM), as well as PCAB to carbonic anhydrase III were used for identification of the cell types in the vas deferens cell culture of guinea pig. Smooth muscle cells (SMC) are identified by intensive staining of PCAB to SMM. Fibroblast-like cells (FBL) are determined by the presence of the filament finest network, apparently responding to the myosin non-muscular forms, which are present in PCAB to SMM. The epithelial cells are stained by MCAB to cytokeratins. PCAB to carbonic anhydrase III interact with all three cell types. In the majority of SMC the enzyme is detected as solitary stripes, though there are diffuse ones across the whole cytoplasms, the nucleus remains clearly visible. Carbonic anhydrase III in epithelial cells is detected only in nucleoli and along nucleus membrane while in FBL--in nucleoli and cytoplasm as focal granulation. PCAB to carbonic anhydrase III may serve as a universal marker for identification of cell type in the guinea pig vas deferens cell culture.     

Binding of adenosine analogs with A1-type purine receptors in the guinea pig ileum

Several N6-adenosine analogs were synthesized and their A1-purine receptor binding affinities were determined. Our results demonstrate that N6-allyl- and N6-aminopropanol-adenosine increase affinities to A1-purine receptor in guinea pig ileum.     

Ontogenetic development of 3,5-adenosine monophosphate phosphodiesterase in guinea pig and rat ileum.

The postnatal development of phosphodiesterases with low and high Michaelis constants in guinea pig and rat ileum was studied. The tow types of phosphodiesterases were found to increase their activity post partum, and this increase was particulary pronounced in the rat. The rise in the enzyme activity took place mainly at the expense of the increase of the phosphodiesterase with high Km. In addition to the quantitative differences in the phosphodiesterase activity of yound and adult animals, considerable age differences were also observed in the sensitivity of the enzyme to inhibitors and activators. These data can contribute to the explanation of the differences in the action of some drugs influencing the phosphodiesterase in young and adult organisms.     

A 45Ca autoradiographic and stereological study of freeze-dried smooth muscle of the guinea pig vas deferens

In an effort to more clearly elucidate the role of cellular structures as calcium sinks and sources in smooth muscle cells, the intracellular distribution of radioactive calcium was evaluated by a new method based on freeze-drying. The guinea pig vas deferens was exposed to a physiological salt solution that contained 45Ca. The muscle was then freeze-dried and prepared for electron microscope autoradiography. The grain density over the plasma membrane, mitochondria, and sarcoplasmic reticulum (SR) was significantly greater than that of the matrix. These results suggest that the plasma membrane, mitochondria and SR have the capacity to accumulate calcium. Which of these structures serve as a source of calcium for contraction remains to be determined. A stereological comparison between freeze-dried and conventionally prepared smooth muscles revealed several differences. The cross- sectional area of freeze-dried cells was about twice that of conventionally prepared cells. Moreover, mitochondria and sub-surface vesicles occupied a significantly smaller percentage of the cell in the freeze-dried tissue than they did in the conventionally prepared tissue.