Palate shelf movement in mouse embryo culture: evidence for skeletal and smooth muscle contractility.

Previous biochemical and morphological studies have shown the presence of contractile proteins in mouse palates at the time of shelf movement. In order to determine whether the palatal contractile proteins function in shelf rotation, an embryo culture system in which palate shelves rotate has been developed. $\text{A}\text{J}$ mouse fetuses with tongues removed (day 14.75) have been cultured close to the time that palatal shelves move in vivo and pharmacological agents added. The anterior end of the palate shelf completely rotated after overnight culture in the presence or absence of drugs. However, rotation of the posterior end of the palate was only partial. Agents that stimulate skeletal and smooth muscle contractility, pyridostigmine (2 × 10^?6–9 × 10^?5M) and bethanechol (10^?10–10^?4M), respectively, both enhanced posterior shelf rotation after overnight culture. Pyridostigmine (9 × 10^?5M) increased posterior shelf rotation 74% over control; bethanechol (10^?4M) 53%. Pyridostigmine effected an appreciable increase in anterior shelf movement within 60 min, while bethanechol stimulated posterior shelf rotation by 60% in that time. These results imply a cholinergic involvement in palate shelf rotation. Furthermore, contraction of “smooth muscle-like” structures previously found on the tongue side extending from top mid-palate to the posterior end may be involved in posterior palate shelf rotation; and contraction of skeletal muscle observed on the oral side posteriorly may aid both posterior and anterior shelf movement.     

Hormonal regulation of macrophage collagenase activity.

Whereas peritoneal macrophages from nonpregnant guinea pigs were stimulated $\text{in vitro}$ by endotoxin to produce collagenase on the second day of culture, those from pregnant guinea pigs were incapable of this response. However, if the cells from pregnant animals were preincubated for one day prior to endotoxin stimulation, collagenase activity could be detected. Injection of either estrogen or progesterone into guinea pigs at doses comparable to those found during pregnancy prior to removal of the peritoneal cells also inhibited the $\text{in vitro}$ stimulation of collagenase production. The addition of these hormones $\text{in vitro}$ revealed that at 5 × 10^?6 M estrogen and progesterone inhibited 53% and 100% respectively of the collagenase activity. Addition of both hormones at a final concentration of 5 × 10^?7 M of each inhibited 87% of the activity indicating a synergistic effect since this concentration of either hormone alone was ineffective.     

Inhibition of spider mite ATPases by plictran and three organochlorine acaricides

The ATPase enzyme system from two-spotted spider mites, $\text{Tetranychus}$$\text{urticae}$ (Koch) was sensitive, in vitro, to four acaricides. Tricyclohexylhydroxytin (Plictran^R) was an outstanding inhibitor of oligomycin-sensitive (mitochondrial) Mg²⁺ATPase from fish brain and spider mite homogenates. The I₅₀ values were 6.6×10^?11M and 6.2×10^?10M, respectively. Less effective were chlorbenside, chlorfenethol and ovotran. Plictran at a higher concentration (2×10^?7M) was also more effective on Na⁺-K⁺ATPase both in mites and fish brain homogenates as compared to chlorfenethol, chlorbenside and ovotran. Plictran inhibited oligomycin-insensitive Mg²⁺ATPase at concentrations of 10^?8M but stimulated at high concentrations (5×10^?6M and higher).     

Catecholamine-mediated constrictor effects of aldosterone on vascular smooth muscle

The possibility that the corticosteroid hormone, aldosterone, might possess direct vasoconstrictor properties was examined in the isolated central ear artery of the New Zealand white rabbit. Aldosterone alone produced only minimal contractile effects in the arterial segments; but following pretreatment of the tissue with desipramine (10^?7M), a blocker of neuronal uptake of norepinephrine, aldosterone concentrations of 10^?6M, 10^?5M, and 10^?4M produced stepwise contractile responses of 0.16 ± 0.03 (SE)g, 0.48 ± .04g, and 1.31 ± 0.06g. The possible involvement of norepinephrine in this action of aldosterone was tested in a series of tissues stored for 2 days at 2°C in Krebsbicarbonate medium so as to deplete endogenous catecholamine stores. Treatment with desipramine followed by aldosterone (10^?4M) now produced an average contraction of only 0.1 ± 0.06g; but if the labile neuronal tissue stores of norepinephrine in these tissues were then replenished by exposure to norepinephrine 10^?7M, contractions of 1.2 ± 0.3g now occurred when desipramine and aldosterone were added. To examine whether aldosterone's action might be due to blockade of extraneuronal norepinephrine uptake (uptake-2), ³H-norepinephrine was added to ear artery tissues exposed to desipramine with or without aldosterone: a significant (P<0.005) decrease of 25% in ³H-norepinephrine uptake occurred in the tissues treated with aldosterone. In further studies, the contractile effects of aldosterone could be prevented by pretreatment with prazosin (10^?7M) or phentolamine (10^?7M); if added after the aldosterone, each of these alpha-blockers completely reversed the contractile responses. Although the physiological relevance of these findings is yet to be fully defined, these studies indicate that the $\text{in}$$\text{vitro}$ contractile effects of aldosterone are dependent upon its inhibition of extraneuronal uptake of endogenous norepinephrine; it is likely that the resulting increase in extracellular norepinephrine concentration then produces vasoconstriction by stimulation of post-synaptic alpha-adrenergic receptors.     

The oviduct musculature of the cockroach Leucophaea maderae and its response to various neurotransmitters and hormones

The musculature of the oviduct consists of an outer, irregular layer of longitudinal muscle and an inner layer of circular muscle. The four basic modes of activity—compression, segmentation, peristalsis, and reverse peristalsis—were evident in the isolated oviduct. These spontaneous events often occurred in an organized sequence. In fact eggs could be transported down the lateral oviducts by this myogenic activity once the sphincter between the common oviduct and vagina was severed. Myographic recordings were made of only the contractions of the longitudinal muscles. L-glutamate caused a distinct phasic contraction at 2.2 × 10^?5 M. The response became larger and more complex as the concentration of the amino acid was increased. Acetylcholine (1.6 × 10^?5 M) caused either a phasic or tonic response, or a combination thereof. By contrast, 5HT and tyramine simply increased the frequency of small phasic contractions, although in some preparations both monoamines caused an inhibition. The ecdysones, a juvenile hormone analogue (1 × 10^?6 M), and prostaglandin E₂ had no effect on oviduct activity. Initially high KCI solutions (162 mM) without Ca⁺⁺ induced a strong contraction but subsequent additions failed to do so. However, when a high KCI solution (158 mM) with 2 mM Ca⁺⁺ was added to the preparation the response was partially restored. Also the potent calcium antagonist Mn⁺⁺ (2mM) can suppress spontaneous activity.     

Stereospecific 3H-nicotine binding to intact and solubilized rat brain membranes and evidence for its noncholinergic nature

³H-nicotine binding was performed on intact and solubilized rat brain membranes as well as membranes from the electric organ of the $\text{Torpedo}$ fish. The K_d for binding to intact and solubilized rat brain membranes was 5.6 × 10^?9 M and 1.1 × 10^?8M respectively, and the binding capacity 2.0 × 10^?14 and 3.0 × 10^?13 moles /mg protein respectively. The K_d for Torpedo membranes was 3.1 × 10^?7M and the binding capacity 6.8 × 10^?13 moles/mg protein. The binding was stereospecific with the affinity of the (?)-nicotine being about 8 times greater than the (+)-nicotine with all three preparations. The relative affinity for the nicotine binding site of nicotinic cholinergic drugs was considerably less in rat brain than in $\text{Torpedo}$ membranes, where the sites are mainly cholinergic. A comparison was made of the ability of a variety of cholinergic drugs and nicotine derivatives to compete with ³H-nicotine binding and their relative pharmacologic potency to produce or inhibit a characteristic prostration syndrome caused by (?)-nicotine administered intraventricularly to rats. From such studies it was concluded that nicotine, in part, may be interacting at noncholinergic sites in rat brain.     

The secretory response in dissociated acini from the rat submandibular gland

Rat submandibular gland was dissociated by enzymatic digestion with collagenase and hyaluronidase, followed by mild mechanical shearing and filtration through a nylon mesh. The dissociated cell populations contained predominantly groups of acinar cells which maintained their acinar arrangement. The morphological and functional viability of the cells was confirmed by electron microscopic examination and a normal secretory response to β-adrenergic or cholinergic stimulation was observed. Both isoproterenol (IPR) and carbachol caused the fusion of secretory granules into large vacuoles which were also continuous with the lumen, and into which the secretory product was released. Secretion was assessed quantitatively from the incorporation of ¹⁴C-glucosamine into the acinar cells and its subsequent release into the culture medium as labelled glycoprotein. IPR stimulated secretion to 125% of untreated controls in the concentration range 5 × 10^?5 to 5 × 10^?7 M, and to 110% of controls at 5 × 10^?8 M, after 40 min incubation. Carbachol stimulated secretion to 131% of controls at 5 × 10^?5 M and to 115% at 5 × 10^?6 M but had no effect at 5 × 10^?7 or 5 × 10^?8 M. The secretory response was blocked by the respective β-adrenergic and cholinergic antagonists, propranolol and atropine. These findings show that dissociated rat submandibular acinar cells provide a useful in vitro model for the study of mucus synthesis and secretion.     

Anomalous enhancing effect of hydroxyurea on DNA synthesis

$\text{In vitro}$ cultures of $\text{Crithidia}$ sp. were exposed to various concentrations of hydroxyurea (HU) during the logarithmic phase. In the presence of 5 × 10^?2M HU, cell division was completely blocked after an initial increase in cell numbers by about 20%. Inhibition of incorporation of ³H-thymidine into acid-insoluble material was effective within 1 hr of exposure to the drug (5 × 10^?2M) and it reached a level of 80% after 8 hr. At lower concentrations (5 × 10^?4M ? 1 × 10^?3M), however, incorporation of ³H-thymidine was remarkably increased while cell division remained unaffected indicating that the increase in incorporation was not due to increased DNA synthesis in preparation for cell division.     

Studies on two isozymes of aconitase from Bacillus cereus T. III. Enzymatic properties.

The present communication describes a comparative study of some enzymatic properties of an early and a late aconitase (EC.4.2.1.3) present in $\text{Bacillus}\text{cereus}$ T cells of 5 and 12 hr culture age, respectively. The activity of both enzymes increased linearly with increase in enzyme concentration. They demonstrated similar pH *7.5) and temperature (30 C) optima, but differed in their activation energy and affinity for substrate. Late aconitase had higher activation energy (16,100 cal) as compared to early aconitase (9,200 cal). Early aconitase showed a Km value of 100 × 10^?4M for sodium citrate and 33.3 × 10^?4M for isocitrate. Late aconitase exhibited 5 to 7 times greater affinity for citrate and isocitrate yielding Km values 14 × 10^?4M and 7 × 10^?4M, respectively. On the basis of available evidence, it is suggested that early and late aconitase present in 5 and 12 hr aged cells of $\text{Bacillus}\text{cereus}$ T behave as isozymes, and may be designated as aconitase (EC.4.2.1.3) isozyme I and aconitase (EC.4.2.1.3) isozyme II, respectively. The significance of their plausible role during growth and sporulation has been discussed.     

Specific antagonism by metoclopramide of dopamine-induced relaxation on isolated rabbit mesenteric arteries contracted with prostaglandin F2alpha.

On isolated rabbit mesenteric arteries pretreated with phenoxybenzamine (10-5M) and contracted with prostaglandin F_2α (PGF_2α) dopamine (10^?6M to 3×10^?4M) and isoprenaline (10-9M to 10-5M) caused a dose-related relaxation. Pindolol (10^?7M) significantly suppressed the effects evoked by isoprenaline, but did not affect those produced by dopamine. The dopamine receptor antagonist metoclopramide (5×10^?5 and 10^?4M), however, shifted the dose-response curve for dopamine-induced relaxation significantly to the right in a concentration dependent manner without affecting relaxations caused by isoprenaline or papaverine. These results demonstrate for the first time a specific antagonism to dopamine-induced relaxation on rabbit mesenteric arteries $\text{in vitro}$. They support the hypothesis of the existence of specific dopamine receptors in vascular smooth muscles.     

A new antibiotic with known resistance factors,G418, inhibits plant cells

Growth rates of two lines of tobacco ($\text{Nicotiana}\text{tabacum}$) cell suspension cultures were measured in the presence or absence of G418, a new 2-deoxystreptamine antibiotic related to Gentamycin. Cell growth rates of $\text{N}$. $\text{tabacum}$ cv. Burley were inhibited at drug concentrations as low as 1.65 × 10^?7 M. At 4 × 10^?7 M, the doubling time was increased from 1.5 days (control) to 2.3 days (treatment). The drug was lethal to cells at 4 × 10^?6 M, and inhibition was irreversible. Cells of $\text{N}$. $\text{tabacum}$ cv. Wisconsin 38 also were inhibited by the drug, although at slightly higher concentrations (ca. 2–5 fold).In view of our findings, G418 and its associated resistance factors could be of great value in plant genetic engineering.     

Vasoactive intestinal polypeptide: increased tone, enhancement of acetylcholine release, and stimulation of adenylate cyclase in intestinal smooth muscle

Vasoactive intestinal polypeptide (VIP) was examined $\text{in vitro}$ for effects on tone and neuronal release mechanisms in intestinal smooth muscle since this is a site of high peptide concentration. VIP contracted the guinea pig ileum and rabbit jejunum in concentrations ranging from 10^?9 to 10^?7 M. Increased tone in the guinea pig ileum was partially antagonized by the anticholinergic agent, atropine (4.38 × 10^?6 M) suggesting that one component of the contractile response was due to the indirect release of acetylcholine. The H₁ receptor antagonist, pyrilamine, did not alter the increased tone produced by VIP indicating that histamine release did not contribute to the ileal contractile response and that VIP exerted a selective effect to enhance neuronal release of acetylcholine. The ability of VIP to modulate acetylcholine release was confirmed in field stimulated ileal preparations where VIP increased the force developed to endogenously released acetylcholine without altering the direct response to acetylcholine. In rabbit jejunum and ileal smooth muscle, VIP related cyclic AMP levels. However, inhibition of phosphodiesterase with papaverine did not potentiate either the VIP-induced ileal contraction or enhancement of the field stimulated response. This raises the possibility that increases in intestinal cyclic AMP may be involved more in VIP-induced alterations in ion transport or secretory phenomenon than in intestinal motility. These studies describing the ability of VIP to modulate acetylcholine release and to increase ileal tone are consistent with the proposed role of VIP in intestinal patholgies involving excessive mucous secretion and motility.     

Bioreduction of nitroxides by Staphylococcus aureus.

Two nitroxide radicals (TEMPO, I; OXAN, II) and a spin labeled penicillin (III) were reduced by $\text{Staphylococcus aureus}$. A short induction period preceded zero order reduction of these substrates leading to a K_m of 8 × 10^?4M, 6.67 × 10^?5M and 5.7 × 10^?4M and V_max of 106, 26 and 11 μ mole/min mg bacteria for I, II and III, respectively.     

Progressive increase with time after sensitization in the functional affinity of T lymphocytes

The dose response relationship in peritoneal cell migration inhibition, elicited by various concentrations of ABA-Tyr, was studied in guinea pigs sensitized with a standard dose of ABA-Tyr. The reactivity to 2 × 10^?7 or 2 × 10^?8 mole/ml appeared at 2 wk, and remained at the maximum level from 4 wk to 8 mo after sensitization. The inhibition by 2 × 10^?9 mole/ml increased up to 4 mo and 2 × 10^?10 mole/ml was first inhibitory at $\text{6}\text{1}\text{2}$ mo. The change in the slope of the dose response curve was a property of the ABA-specific cells. As there is no B-cell response to ABA-Tyr, the finding shows that the functional affinity of specific T cells progressively increases with time after sensitization.     

Stereospecific interaction of opiate narcotics in binding of 3H-dihydromorphine to membranes of rat brain

Membranes from homogenates of corpus striatum bound ³H-dihydromorphine in a saturable fashion with a Km value of 1 × 10^?9M. The binding of ³H-dihydromorphine to the membranes was reduced to about 10% by 10^?7M levorphanol but not by 10^?7M dextrorphan. The binding of ³H-dihydromorphine became less sensitive to 10^?7M levorphanol when the concentration of ³H-dihydromorphine was greater than 2 × 10^?9M. Other opiate narcotics, e.g. morphine and $\text{l}$-methadone, were as effective as levorphanol in competition for the binding ³H-dihydromorphine with ED₅₀ values of 2–4 × 10^?9M. $\text{d}$-Methadone and dextrorphan were about 1/50 and 1/2000 as effective as their respective levo-isomers. The opiate antagonist, naloxone, also competed effectively for the binding sites with an ED₅₀ value of 3.3 × 10^?9M. Substances like acetylcholine, choline, serotonin, norepinephrine and dopamine were ineffective. Only ionophores specific for divalent cations stimulated the binding of ³H-dihydromorphine suggesting that some endogenous divalent cations may be inhibitory to the binding of the opiate narcotic. The receptors of ³H-dihydromorphine probably exist in the membranes of nerve endings and have a density of 6 × 10¹² sites per g in corpus striatum. We conclude that the described technique can successfully detect the opiate narcotic receptors in the central nervous system without the usual method of displacement.     

Relative contracting adn relaxing potencies of a series of prostaglandins on isolated canine mesenteric artery strips

The contracting and relaxing potencies of anf interactions between a number of prostaglandins (PGs) were studied $\text{in vitro}$ on spiral strips of small canine mesenteric arteries (outside diameter < mm). PGF₂α and PGE₂, the most potent contracting PGs, were nearly equal in potency (EC₅₀ 4 × 10^?7M) and did not cause relaxation under our experimental conditions. PGI₂ and PGE₁ were equal and the most potent relaxing PGs (EC₅₀ 3 × 10^?9M). PGE₁ also caused contraction, but this effect was not consistent. PGI₂ did not cause contraction in concentrations up to 3 × 10^?6M. In higher concentrations, however, it caused abrupt and near maximal contraction. PGD₂ was weak in both respect, causing incomplete relaxation and contraction or biphasic effects. Interaction studies showed that PGE₁ and PGI₂ mutually excluded the relaxing effects of each other. PGE₁ also reversed the relaxing effect of isoproterenol. However, pre-exposure to PGD₂ did not attenuate the relaxing effect of PGE₁ or PGI₂ nor was the relaxing effect of PGD₂ changed by pre-exposure to PGE₁. Two different orders of potency of PGs suggest two PG receptors subserving contraction and relaxation, respectively. Further, it appears that several PGs can act upon both receptors which may explain unusual interactions between the PGs and some of their atypical effects. Finally, the data also suggest that there may be subtypes of the PG receptors subserving contraction and relaxation.     

Mechaism of Arthrobacter sialophilus neuraminidase: The binding of substrates and transition-state analogs

2-Deoxy-2,3-dehydro-N-acetylneuraminic acid and its methyl ester are competitive inhibitors of Arthrobacter sialophilus neuraminidase with K_i = 1.4 × 10^?6$\text{M}$ and 4.8 × 10^?5$\text{M}$, respectively. The K_m for the substrate, N-acetylneuraminlactose, is 1.0 × 10^?3$\text{M}$. These data, taken together with the conformation of these compounds, indicate that these compounds are transition-state analogs of the enzyme. These results also suggest that the substrate upon binding to neuraminidase is distorted to a conformation approaching that of a half-chair.     

A serotonin sensitive guanylate cyclase associated with specific neurotransmitter binding sites on isolated synaptic membranes from mature rat brain.

Results from this study indicate that adult rat brain posesses guanylate cyclase activity sensitive to serotonin (5-HT) and localized in the synaptic plasma membrane. The enzyme appears to have multiple activation sites for 5-HT with specific activity maxima at the 5-HT concentrations of 5 × 10^?10M and 7 × 10^?8M respectively. The rates of guanosine-3′:5′-monophosphate (cyclic GMP) formation at these concentrations of 5-HT are, respectively, 170% and 307% above the endogenous or basal production rate of 2.7±0.3picomoles/minute/milligram of synaptosomal membrane protein. We have also been able to identify $\text{four}$ distinct types (Type #1, #2, #3, and #4) of high affinity, specific binding sites for 5-HT on isolated synaptosomal membranes from rat brain. Dissociation constants of 2.6 × 10^?10M, 2.5 × 10^?9M, 7.0 × 10^?9M, and 4.6 × 10^?8M, characterize the binding of 5-HT to our sites of Type #1 through Type #4 respectively. The specific, high affinity binding was saturated at 5-HT concentrations of 5 × 10^?10M for the Type #1 sites, 5 × 10^?9M for our Type #2 sites, 1 × 10^?8M for our Type #3 sites, and 7 × 10^?8M for our Type #4 sites. The 5-HT concentrations producing saturation of our specific binding sites of Type #1 and Type #4 are virtually identical to those that elicit the two maxima of 5-HT stimulated cyclic GMP production, indicating that a membrane-bound guanylase cyclase may be closely associated with certain 5-HT receptors and/or re-uptake sites.     

Halogenated biphenyls as AHH inducers: Effects of different halogen substituents

4′-Iodo-, 4′-bromo-, 4′-chloro- and 4′-fluoro-2,3,4,5-tetrachlorobiphenyl were administered to immature male Wistar rats and the effects of this homologous series of 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal drug-metabolizing enzymes were determined. All the halogenated biphenyls increased microsomal benzo[a]pyrene hydroxylase (or aryl hydrocarbon hydroxylase, AHH), ethoxyresorufin (ER) O-deethylase and dimethylaminoantipyrine (DMAP) N-demethylase. The effects of the 4′-halo-2,3,4,5-tetrachlorobiphenyls on the microsomal enzyme activities and on the relative peak intensities and spectral shifts of the reduced cytochrome P-450:CO and ethylisocyanide (EIC) binding difference spectra were similar to those observed after coadministration of phenobarbitone (PB) and 3-methylcholanthrene (MC). The relative activities of the halogenated biphenyls were determined using two $\text{in}$$\text{vitro}$ assays; namely cytochrome P-448 associated induction in rat hepatoma H-4-II E cells in culture and competitive binding to the hepatic cytosolic $\text{Ah}$ receptor protein from male Wistar rats. Dose-response experiments for the iodo, bromo, chloro and fluoro analogs gave EC₅₀(M) values of 8.5×10^?9, 6.6×10^?8, 5.7×10^?7, and 3.3×10^?5, and 1.5×10^?6, 2.5×10^?6, 4.1×10^?6 and 2.5×10^?5 for the ER O-deethylase induction and receptor binding assays respectively. The relative potencies of the 4′-halo-2,3,4,5-tetrachlorobiphenyls followed the order I>Br>Cl>F for both assays and differences in the EC₅₀ values for the iodo and fluoro analogs were greater than three orders of magnitude for ER O-deethylase induction in rat hepatoma cells in culture. One possible explanation for these effects may be associated with differences in the polarizability of the laterally substituted halogen groups. However, other differences in the physico-chemical properties of the halogen atoms may also be important.     

The artifactual nature of fluoride inhibition of reverse transcriptase and associated ribonuclease H

Surprisingly, the $\text{sn}$-1 configuration of 1-0-hexadecyl-2-acetyl-glycerylphosphorylcholine showed significant activity, 3.22 × 10^?9 M, when compared to the $\text{sn}$-3 enantiomer, 2.92 × 10^?10 M and a racemic mixture with a value of 7.2 × 10^?10 M. A methoxy substitution at the C-1 or C-2 position of octadecyl glycerylphosphorylcholine gave a derivative with high biological activity for stimulating serotonin release from rabbit platelets. A 1-0-dodecyl-2-methoxy analogue showed very low activity; also, a comparable series of 0-benzyl derivatives were inactive. Examination of 1-0-hexadecyl, 1-0-octadecyl- or 1-0-dodecyl-2-acetyl-$\text{sn}$-glyceryl-3-phosphorylcholine showed that the hexadecyl compound had three times the biological activity of the octadecyl and five times that of the dodecyl.