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Persons with Down's syndrome (DS) are susceptible to severe and precocious periodontal disease. Several organisms have been implicated in the etiology of periodontal disease, including Actinobacillus actinomycetemcomitans (Aa). It is unknown whether circulating antibodies correlate with the severity of periodontal disease in DS. This study determined the circulating antibody titers to Aa in sera of DS and normal patients. Eleven DS patients with periodontal disease (pocket depth > 4 mm), five DS patients with gingivitis (inflammation and pocket depth < or = 3 mm), and 10 non-DS healthy subjects had blood drawn and analyzed for antibody response to Aa. Conventional enzyme-linked immunosorbent assay (ELISA) with goat anti-human IgG was performed for Aa-reactive serum diluted 1:200 to 1:12,800. Geometric mean titer was calculated, and significant differences were noted between the control group and the DS groups (p = 0.05), with the DS periodontal group having the highest response, followed by the DS gingivitis and normal controls, respectively. The DS groups were not significantly different. DS patients may exhibit a higher Aa antibody response, due to the presence of the organism, previous exposure, or in proportion to the extent of their periodontal condition.

An apparatus was developed to apply positive or negative hydrostatic pressure dynamically to periodontal ligament (PDL) cells in vitro. The objective of this investigation was to construct this apparatus and to determine its effects on PDL cells. Human PDL cells were collected from freshly extracted premolars. At the sixth passage, the cells were mechanically stimulated by this apparatus at different magnitudes of continuous positive or negative hydrostatic pressures (PHP or NHP, respectively). The application of PHP between 0.3 and 30 gm/cm2 significantly enhanced prostaglandin E (PGE) production and intracellular cyclic AMP (cAMP) of the cells. In contrast, perturbation by NHP significantly decreased PGE production and intracellular level of cAMP. Proliferation rate increased significantly at 24 and 48 hours due to stimulation of these cells with -30 gm/cm2 of NHP. Challenging these cells with +30 gm/cm2 of PHP significantly decreased the proliferation rate of these cells at 24 and 48 hours. Stimulation by PHP between +30 to +600 gm/cm2 increased cell length and width and appeared to increase surface area attachment to the bottom of the culture dishes. In contrast, NHP (between -30 and -600 gm/cm2) decreased these dimensions and appeared to reduce the surface area of attachment. These results indicate that this type of mechanical perturbation of PDL cells produces physiologic responses and is not detrimental to their vitality.

The objective of this study was to determine how fetal effects are altered when nicotine (N) and caffeine (CA) are administered concurrently at dosages that individually produce minimal effects to the fetus. Female ICR mice were bred overnight and were assigned to four groups: CA (125 mg/kg), N (12mg/kg), CA plus N (125 mg/kg plus 12 mg/kg, respectively) treated, and control (distilled water) groups. Dams were intubated with these dosages three times daily during gestational days (GD) 6-18 and were euthanized on GD 18. Live fetuses were sexed, weighed, and examined for external malformations. One-half of the fetuses were fixed in 10% formalin and examined for internal malformations using Wilson's method. The remaining half was fixed in 95% ethanol (ETOH), stained, and cleared (Inouye's method) for skeletal examinations. Ossification was assessed by staging and measuring craniofacial bones, and counting ossification centra in sternbrae and in cervical and sacrococcygeal vertebrae. Data were analyzed by analysis of variance (ANOVA) followed by Student-Newman-Keuls post-hoc tests set at p < .05 significance level. The litter was used as the unit of measure and the ANOVA main effects were CA, N, and an interaction term (CA+N). In comparison to controls, CA treatment resulted in reduced bone measurements or reduced ossification scores in 5 of the 19 parameters examined, whereas for N only five parameters were significant. The main effects for interaction of CA+N were significant for seven parameters measured. Although it is difficult to assign the specific type of drug interaction that occurred because results were not completely consistent for all parameters measured, it may be concluded that in most parameters measured both CA and CA+N were different from controls, but CA was not different from CA+N. Under the experimental conditions of this study, we found that of the two drugs, caffeine had a significantly greater effect on fetal growth and ossification than nicotine.

Studies have shown that patients undergoing orthodontic tooth movement can experience varying degrees of discomfort. The objective of this study was to determine whether nonsteroidal antiinflammatory agents, such as aspirin or ibuprofen, which inhibit prostaglandin synthesis through acetylation and inactivation of the enzyme cyclooxygenase, can suppress orthodontic discomfort by inhibiting the inflammatory response normally observed after orthodontic adjustments. A total of 77 patients were included in a double-blind, randomized, parallel, placebo-controlled, single-dose, analgesic efficacy evaluation of ibuprofen and aspirin. Patients were divided into three groups. Group A received one dose of the drug ibuprofen (400 mg), group B received aspirin (650 mg), and group C received a placebo (beta-lactose). The level of discomfort was assessed using a visual analogue scale at 2, 6, and 24 hours and 2, 3, and 7 days after the insertion of either orthodontic separators or an initial arch wire. A repeated measures analysis of variance and post hoc studentized range statistics showed that the placebo group had significantly more discomfort than either the ibuprofen or the aspirin group at all the time intervals tested. In addition, ibuprofen produced significantly less discomfort than aspirin at the 6 and 24-hour and 2-day time phase after separator placement; and 2 and 6 hours and 2, 3, and 7 days after arch wire placement. These results support a recommendation for ibuprofen as a preferred analgesic in the treatment of discomfort because of postorthodontic adjustments.

Three dosages of Smokeless Tobacco (ST) extract were given to pregnant Sprague-Dawley rats by oral gavage on gestational days (GD) 6-20. The three dosages contained ST extract equivalent to 1.33 mg/kg nicotine (STD-1), 4.0 mg/kg nicotine (STD-2), and 6.0 mg/kg nicotine (STD-3). Dams were intubated three times per day at 8 a.m., 11 a.m., and 2 p.m., providing total daily ST dosages of 4 mg/kg, 12 mg/kg, and 18 mg/kg, respectively. Controls received equivalent volumes of water by gavage. Dams were allowed to deliver, and all biological mothers raised their own pups. On postnatal day 1 (PND 1), litters were culled to 4 +/- 1 females and 4 +/- 1 males. Weights, physical landmark development, and behavioral performance of pups were monitored during pre- and post-weaning periods. Behavioral tests included surface righting, negative geotaxis, swimming development, open field activity, and active avoidance in shuttle box. Our results show that the two higher doses resulted in reduced maternal weight gain. During the pre-weaning period, significant pup weight reductions were noted in the STD-2 pups until PND6, and in the STD-3 group until PND15. In the STD-1 group no statistically significant weight reduction was noted on PNDs 1 and 3, but starting with PND6, pup weights surpassed control group weights. This weight difference persisted throughout the post-weaning period also (P < .05 on PND30 and PND42). The STD-3 pup weights continued to be consistently and significantly (P < .05) reduced throughout the post-weaning period (except on PND24); likewise, the STD-2 pups continued to have lower weights, but at a significant level (P < .05) on PND30 only. The incidence of deaths was increased in a dose-related manner. No significant differences were noted for pinna detachment and incisor eruption; however ST-treatment was significant in affecting earlier eye opening and vaginal patency. N significant ST treatment effects were seen on negative geotaxis, but for surface righting a decreased success rate was noted for the ST-treated groups. Significant differences were noted in swimming development, with the STD-2 pups performing best. Open field activity, as expected, increased from the pre-weaning to post-weaning periods. During the pre-weaning period the STD-3 pups were more active, and during post-weaning the STD-1 pups were more active, but no differences were noted in vertical activity or in the number of stereotypical movements. No treatment-related differences were noted in the active avoidance shuttle box.(ABSTRACT TRUNCATED AT 400 WORDS)

The objective of this study was to examine the effect of an aqueous extract of smokeless tobacco (ST) on the development and bone ossification of the Sprague Dawley rat fetus at known nicotine blood levels. Dams were intubated with the ST extract three times daily on gestational days (GD) 6-18 with one of the following: ST equivalent to either 1.33 mg nicotine/kg body weight (STD-1), 6 mg nicotine/kg body weight (STD-2), or equal amounts of distilled water (sham-treated controls). Parallel groups of rats were used for nicotine-blood-level determinations. Dams were killed on GD 19, fetuses and placentas were weighed, and resorptions, deaths, and/or malformations were noted. Two thirds of the fetuses were further examined by Wilson's method, and the remaining one third was stained and cleared for skeletal examinations. Mean plasma nicotine levels, determined in a parallel group of nonpregnant/pregnant rats, were 220.4/283.3 ng/ml in the STD-1 group and 869.1/846.3 ng/ml in the STD-2 group. At these ST dosages, weight gain of dams was reduced in comparison with sham-treated controls (P < .05), but fetal weights were reduced in the STD-2 group only. Placental weights, litter size, resorptions, deaths, and malformations were not significantly affected. Skeletal examinations revealed several dose-related differences between the ST-treated and sham-treated control groups. In the STD-1 group, reductions in ossification were seen in the nasal and femur width measurements only. In the STD-2 group, reductions in ossification were seen in femur length and width, in the number of ossification centers in the forelimb, in the maxillary, mandibular, and nasal bone measurements. We conclude that under these experimental conditions the effects of ST at the low dose are minimal, whereas the high ST dose resulted in significant growth retardation and decreased ossification levels (P < .05).

Two dosages of Smokeless Tobacco (ST) extract were given to gravid Sprague-Dawley rats by oral gavage on gestational days (GD) 6-20. The low dosage contained ST extract equivalent to 1.33 mg/kg nicotine (STD-1), and the high dosage contained ST extract equivalent to 4.0 mg/kg nicotine (STD-2). Dams were dosed three times daily at 8 a.m., 11 a.m., and 2 p.m., thus providing total daily nicotine equivalent dosages of 4 mg/kg/day and 12 mg/kg/day. Controls received equivalent amounts of distilled water by gavage. Dams were allowed to deliver and all experimental pups were fostered to control mothers. On postnatal day 1 (PND 1) litters were culled to 4 +/- 1 females and 4 +/- 1 males. Weights, physical landmark development, and behavioral performance of pups were monitored during pre- and post-weaning periods. Behavioral tests included: surface righting, negative geotaxis, swimming development, open-field activity, active avoidance in shuttle box, and Cincinnati swimming maze. Our results show that the STD-2 dose resulted in reduced maternal weight gain. Offspring weights were reduced in a dose-related manner, with the most consistent weight deficits seen in the STD-2 group until PND29. Consistent STD-1 weight deficits were seen up to PND 8. The incidence of deaths was increased in the STD-2 dosage group. No significant treatment-related differences were observed in development of physical landmarks. Male STD-2 pups righted faster than controls, and significant differences were noted in swimming development with the STD-1 group of pups performing less effectively than controls. Activity levels, assessed during both pre- and post-weaning periods were not affected. No treatment-related differences were seen in the active avoidance shuttle box or Cincinnati swimming maze tests, which assessed learning. Female brain weights were reduced in the STD-1 treatment group.

Tobacco products and alcohol are commonly used as nonmedicinal drugs by pregnant women, and both are known to cause various effects on the fetus and the newborn. The objective of this study was to examine the fetal effects of both drugs when administered individually and simultaneously to pregnant CD-1 mice at moderate dosages. Specifically, we wanted to determine whether or not the effect on the fetus of these two biologically active substances was additive, ameliorative, or synergistic. A total of 65 CD-1 dams were divided among four groups receiving either ST equivalent to 8 mg/kg nicotine, ethanol (ETOH) 1.8 g/kg, a combination of ST+ETOH in the same dosages, or D-glucose (controls and ST alone) to supply calories equivalent to the dose of ethanol. Mice were dosed three times per day on gestational days 6-15. On gestational day 17 all dams were killed, fetal and placental weights recorded, and the number of resorbed, dead, and malformed fetuses noted. The mean maternal plasma drug levels were: nicotine-321 ng/ml and ethanol-0.105 g%. No significant differences were observed in maternal weight gain, litter size, or in the incidence of resorptions, deaths and/or malformations. Fetal weights were reduced in all three treatment groups (P less than 0.05), with the greatest reduction (13% decrease) recorded in the ST group, followed by a 9% decrease in the ETOH group, and a 7% decrease in the ST+ETOH group. Placentas of the ST group weighed significantly less (P less than 0.05) than controls. Ossification of the fetal skeleton, observed in ten sites, was affected to the greatest extent in the ST group, followed by the ETOH and ST+ETOH groups. Craniofacial measurements were significantly affected (P less than 0.05) in all three treatment groups, compared to controls. We conclude that under these experimental conditions, in terms of fetal growth and ossification, ST had the greatest effect, followed by ETOH and ST+ETOH. The interaction of ST+ETOH was neither additive, synergistic, nor ameliorative.

Cytokines are local mediators released by cells of the immune system in response to stimulation by a variety of agents. These polypeptides may interact directly or indirectly with bone cells. The objectives of this study were (1) to localize prostaglandin E (PGE) and the cytokine interleukin-1 beta (IL-1 beta) in the periodontal ligament after the application of mechanical force to teeth in vivo and (2) to determine the effects of mechanical stress or IL-1 beta (or the two in combination) on PGE synthesis and bone resorption by fibroblasts in the human periodontal ligament (PDL). In 24 female cats, one maxillary canine was tipped distally by 80 gm force for 12 hours, 24 hours, or 7 days. PGE and IL-1 beta were localized immunohistochemically in serial jaw sections, and semiquantitation of cellular-staining intensity was done by microphotometry. Unstressed periodontal ligament cells stained mildly for PGE and IL-1 beta, but the staining intensity increased significantly in sites of tension. Human periodontal ligament fibroblasts were preincubated with mechanical stress and/or IL-1 beta in the presence or absence of indomethacin for 1 hour. Then the media were replaced by BGJb (Fitton-Jackson modification) medium (GIBCO), and incubation was continued for 4, 8, or 24 hours in conditioned media. PGE concentrations in conditioned media were determined by radioimmunoassay, and bone-resorbing activity in conditioned media was assessed by 45Ca release from prelabeled neonatal mouse calvaria. The conditioned media derived from cells stimulated by mechanical stress plus IL-1 beta caused significantly more bone resorption than the conditioned media obtained from cells that had been treated by each factor alone. The addition of indomethacin did not inhibit bone resorption completely. These results demonstrate that periodontal ligament cells respond to mechanical stress by increased production of PGE, and that IL-1 beta enhances this response.

The objective of this study was to examine the effect of subchronic administration of an aqueous extract of smokeless tobacco (ST) on the development of the CD-1 mouse fetus. Mice were administered ST for approximately 5 weeks: for 2 weeks prior to breeding, during breeding, and during gestational days 0 to 17. Thus the initial peak nicotine levels occurred prior to breeding and not during the critical periods of gestation. Two ST dosages were administered by gastric intubation three times daily: ST/D-1, equivalent to a dose of 12 mg nicotine/kg of body weight, and ST/D-2, equivalent to 20 mg nicotine/kg body weight. Maternal plasma nicotine levels were determined 30 min after the second intubation during the pretreatment and gestational phases of treatment. At these ST dosages, the weight gain of ST-treated dams was not significantly affected in comparison to treated controls. The mean maternal plasma nicotine level for the low-dosage group was 363 ng/ml, and 481 ng/ml for the high-dosage group, with maternal lethality observed at 9.6% and 28.2%, respectively. No significant differences were seen between control and ST/D-1 maternal and/or fetal values, except for placental weights which were heaviest in the ST group (P less than 0.05). Several differences were noted between the ST/D-2 group and controls: fetal weights were reduced by 5.4% (P less than 0.05); decreased ossification was seen in femur measurements and in nine of ten characteristics measured (P less than 0.05); the frequency of resorptions (7.6%) was almost doubled (controls 4.2%); and the frequency of deaths and malformations was not affected. Under these experimental conditions, the low dose produced a negligible effect on the CD-1 mouse fetus and the dam. The high dose demonstrated growth retardation (P less than 0.05), increased embryotoxicity, and a significant decrease in ossification (P less than 0.05).

Human periodontal ligament (PDL) fibroblasts, cultured from extracted healthy premolars, and a cloned osteogenic cell line (MC3T3-E1) were used in this study to determine the effect of intermittent pressure on bone resorption. Cells (1 x 10(5] were incubated with BGJb medium in the presence or absence of the following factors: intermittent negative (-30 g/cm2) or positive (30 g/cm2) hydrostatic pressure and interleukin-1 beta (IL-1 beta, 1 ng/mL), for 24 h. Conditioned media (CM) generated from cultures of either cell types were used for prostaglandin E (PGE) assay, bone resorption assay, and assessment of osteoclast (OC)-like cell formation. Unstimulated PDL fibroblasts or MC3T3-E1 cells produced measurable amounts of PGE and bone-resorbing activity as measured by 45Ca released from mouse calvaria and OC-like cells. IL-1 beta-treated cells showed significantly elevated levels of PGE, bone resorption, and OC-like cell formation, as compared with unstimulated cells. Intermittent positive pressure (IPP) alone stimulated PGE production, but the resultant CM did not stimulate bone resorption or OC-like cell formation when IPP was applied to either cell type. The application of IPP, together with IL-1 beta in CM, caused a slight increase in the number of alpha-like cells, as compared with that of IL-1 beta-treated CM in both cell types. On the other hand, direct application of IPP on mouse bone-marrow cultures significantly increased the number of OC-like cells. This effect was additive in combination with either CM from unstimulated cells or exogenous addition of PGE2.(ABSTRACT TRUNCATED AT 250 WORDS)

Human periodontal ligament (PDL) cells were derived from healthy premolars extracted for orthodontic treatment and were utilized for in vitro experiments in passages 4-6. Human PDL cells were seeded in tissue culture tubes and incubated with interleukin-1 alpha (IL-1 alpha), IL-1 beta, tumor necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), indomethacin, parathyroid hormone (PTH), or their combinations, for 1 h. The medium was then replaced with serum-free BGJb medium and incubated for 24 h without further additions. Prostaglandin E (PGE) concentrations in the conditioned media (CM) were measured by radioimmunoassay, and bone-resorbing activity was measured using 45Ca-labeled neonatal mouse calvariae. The results of this study indicated that (1) unstimulated cultured PDL cells produced PGE, and PDL CM stimulated bone resorption; (2) cytokine-treated (IL-1 alpha, IL-1 beta, and TNF-alpha) PDL cells had increased production of PGE and bone-resorbing activity compared to unstimulated PDL cells; (3) indomethacin completely inhibited PGE production from unstimulated PDL cells but only partially inhibited bone-resorbing activity, indicating that PDL cells produced nonprostaglandin bone-resorbing factor(s); (4) IFN-gamma did not change PGE or bone-resorbing activity production by cytokine-stimulated PDL cells; and (5) PTH treatment of PDL cells in addition to cytokines (IL-1 alpha, IL-1 beta, and TNF-alpha) had additive effects on the production of bone-resorbing activity and synergistic effects on PGE production compared to cytokine treatment alone.

Mononuclear cell production of cytokines that stimulate fibroblast production of prostaglandin E (PGE) is an important mechanism by which mononuclear cells regulate fibroblast function. The objective of this investigation was to determine the effects of the cytokines interleukin 1 beta (IL-1 beta), interleukin 1 alpha (IL-1 alpha), tumor necrosis factor alpha (TNF-alpha), and interferon gamma (IFN-gamma), alone or in paired combinations, on PGE production by near-confluent human periodontal ligament (PDL) fibroblasts in vitro. Premolars extracted in the course of orthodontic treatment were used for this study. Fibroblast cultures, free of epithelial cells, were obtained after the fourth subculture by the use of accurately-timed trypsin treatment. Cells in the fourth to sixth passage, incubated in DMEM supplemented with 10% equine serum, were used for these experiments. Cells (1 x 10(5)) were seeded in 12- x -75-mm tissue culture tubes and incubated with various doses of IL-1 beta, IL-1 alpha, TNF-alpha, and IFN-gamma, alone or in specific combinations, for 15 min, two, 12, 24, and 72 h. PGE concentrations in the media were measured by radio-immunoassay. The results showed that human PDL fibroblasts responded to the administration of cytokines by an elevation in the synthesis of PGE in a dose- and time-related fashion. The increase in PGE production was inhibited by the addition of indomethacin. The interactions between these cytokines varied in degree, depending on the particular combinations of cytokines. In addition, the administration of cytokine combinations was found to be additive, synergistic, subtractive, or suppressive on the production of PGE by PDL fibroblasts, depending on the duration of incubation. These experiments demonstrate the importance of the consideration of the interplay between cytokines produced by mononuclear cells on the mechanisms that regulate the functions of PDL fibroblasts.

Human periodontal ligament fibroblasts and a cloned osteogenic cell line (MC3T3-E1) were seeded (4 x 10(5) cells) on 60 mm Petriperm dishes, which have a flexible plastic growth surface. Cells were stretched by placing the dish on top of a spheroidal convex template, equilibrated to 37 degrees C. The amount of stretch was varied by changing the curvature of the template and calculated as percentage stretch. Both types of cell responded to mechanical stress by elevated synthesis of PGE and cAMP; the addition of interleukin-1 beta to mechanically stretched cells produced further elevation. Synergism between mechanical stress and interleukin-1 beta was found at certain lengths of incubation. The production of cAMP was secondary and dependent on the newly synthesized PGE, as shown in the presence of indomethacin. The two cell types were also different in terms of the timing of their response to mechanical stress and interleukin-1 beta. In the absence of stimuli, periodontal fibroblasts tended to produce PGE continually over time, whereas the MC3T3-E1 cells did not. However, both cell types had elevated PGE levels in response to the stimuli used in this experiment. Periodontal fibroblasts responded to mechanical stress and interleukin-1 beta with significant elevations of PGE as early as 15 min, whereas the MC3T3-E1 cells required 2 h to produce significant elevations for mechanical stress and 15 min for interleukin-1 beta. These findings indicate that the chemical and mechanical signals on these cells are mediated by surface receptors. Locally produced autocrine or paracrine factors can modify the effect of mechanical stress on periodontal and bone cells via the cAMP pathway.(ABSTRACT TRUNCATED AT 250 WORDS)

Cultured human periodontal ligament fibroblasts showed synergistic elevations in the synthesis of prostaglandin E and production of cAMP by the administration of parathyroid hormone and cytokines (interleukin 1 alpha, -1 beta, or tumour necrosis factor-alpha). Unstimulated conditioned media derived from these fibroblasts contained bone-resorbing activity. In addition, conditioned media generated by cytokine-or parathyroid hormone-treated fibroblasts showed further increases in bone-resorbing activity. The effects were additive when the hormone was combined with either one of the cytokines in stimulating bone resorption. These findings suggest that the effect of parathyroid hormones and cytokines together on bone resorption can be mediated in part by human periodontal ligament fibroblasts via PGE production and subsequent PGE action on the osteoclasts.

The stimulation of PGE synthesis and cAMP production by cytokines have important physiological effects in many target tissues. The effects of interleukin-1 alpha and -1 beta, tumour necrosis factor-alpha and interferon-gamma on PGE and cAMP production by periodontal ligament fibroblasts were studied. Fibroblasts in the 4th-6th passage, grown and maintained in DMEM supplemented with 10% equine serum, were incubated with graded doses of the various cytokines for 0.25, 0.5, 1.2, 4, 24, 48 or 72 h. At the end of each incubation, PGE in the medium and the cellular content of cAMP were evaluated by a combined immunohistochemical microphotometric procedure, and conventional radiometric assays. The fibroblasts responded to all the cytokines with a dose- and time-related increase in the levels of PGE and cAMP. Such increases were inhibited by the inclusion of indomethacin in the medium. The addition of exogenous PGE reversed that inhibition in respect of cAMP production. Immunohistochemical localization showed PGE predominantly in the cytoplasm and cAMP in the nucleus. These findings indicate that: (1) human periodontal ligament fibroblasts respond to these cytokines by increased synthesis of PGE and the production of cAMP; and (2) the cAMP production is secondary to the PGE synthesis. They suggest that these cytokines may regulate the function of these fibroblasts in physiological remodelling of the periodontium, as well as in inflammatory reactions.

This study correlates endogenous levels of cAMP and cGMP with their immunohistochemical localization during chondrogenic differentiation of C57B1/6J mouse limb mesenchyme in vivo and in vitro. A transient decrease in cGMP but not cAMP was found from days 12 to 13 in vivo correlating with early stages of chondrogenesis in the developing limb. Intracellular levels of both cAMP and cGMP in high density limb mesenchyme cultures increased 25% after 24 hr in culture when aggregate and nodule formation was detectable. When cells were seeded at different initial plating densities to delay the onset of aggregate and nodule formation, increased levels of intracellular cAMP correlated temporally with the appearance of nodules. Both cyclic AMP and cGMP were immunohistochemically localized in perichondrial cells and chondrocytes in vivo and in vitro. Therefore, (1) cAMP levels correlated temporally with the appearance of chondrogenic cells and (2) cAMP and cGMP were immunohistochemically localized to chondrogenic cells. These data indicate that fluctuations of both cAMP and cGMP levels may be involved in limb cartilage differentiation. Although increases in both nucleotides were found to correlate with the onset of chondrogenesis in vitro, in vivo data suggest that the amount of cAMP relative to cGMP rather than the absolute amount of an individual cyclic nucleotide may be more significant in modulating differentiation.

The objective of this study was to examine the effect of an aqueous extract of smokeless tobacco (ST) on the development of the CD-1 mouse fetus. Three ST dosages were administered three times daily by gastric intubation during gestational days 1-17: 1 X ST equivalent to a dose of 4 mg nicotine/kg body weight, 3 X ST equivalent to 12 mg nicotine, and 5 X ST equivalent to 20 mg nicotine/kg body weight. Maternal plasma nicotine levels were determined 30 minutes after the second daily intubation at five different times during the gestational period. At these ST dosages, the weight gain of ST-treated dams was not significantly affected in comparison to treated controls, though the difference was significant (P less than .05) in comparison to untreated controls. The mean maternal plasma nicotine level for the low dosage (1 X) group was 99.0 ng/ml, which reasonably approximates human consumption levels. The 3 X ST and 5 X ST dosages produced higher nicotine plasma values, 398 ng/ml and 623 ng/ml, respectively, were considerably more toxic to the dams, and resulted in 18% and 31% maternal deaths. Fetal weights were reduced by 7.4% (P less than .001) in the highest ST dosage group (5 X), whereas at the 1 X and 3 X dosages fetal weight differences were not significantly different from treated controls. Resorptions increased in a dose-related manner (P less than .05), ranging from 4.7% in the 1 X, to 6.4% in the 3 X and 8.9% in the 5 X dosage compared to 3.2% in treated controls. External malformations were few and minor in extent. Internal malformations increased in a linear, dose-related manner (P less than .05). Placental weights were unaffected by ST. The results of skeletal examinations were inconclusive. Precocious ossification was seen in 60% and 70% of the parameters measured in the 1 X and 3 X dosage groups, respectively, in comparison to controls. In the 5 X ST group ossification levels were less than in controls for 30% of the parameters measured. Under these experimental conditions the lowest ST dosage (1 X) produced a negligible effect on the CD-1 mouse fetus and the dam. The highest ST dose (5 X) demonstrated embryotoxicity, growth retardation, few malformations, and maternal toxicity. The intermediate dose (3 X) showed a range of effects between the highest and lowest doses to both the fetus and the dam.

Prostaglandins E2 and F2 alpha (PGE2 and PGF2 alpha) have been shown to cause changes in adenosine 3',5'-cyclic monophosphate (cAMP) levels in a wide variety of tissues. In particular, murine palatal mesenchyme responds to PGE2 stimulation with dose-dependent increases in intracellular cAMP levels. These same mesenchymal cells also synthesize PGE2 and PGF2 alpha. The purpose of this study is to localize PGE and PGF2 alpha in the developing murine palate by using immunohistochemical techniques. Fresh frozen cryostat sections of murine C57BL/6J embryo palates (days 12-14 of gestation) were incubated with anti-PGE or PGF2 alpha monoclonal antibodies. On day 12 of gestation, PGE and PGF2 alpha, identified as 3',3-diaminobenzidine (DAB) reaction products, were localized throughout palatal mesenchyme and epithelium; on day 13 of gestation, reaction product indicative of both PGE and PGF2 alpha was detectable primarily in mesenchyme subjacent to palatal epithelium. Extracellular spaces of the adjacent mesenchyme in the central region of the day 13 palate exhibited less reaction product. Palatal epithelium, particularly the medial edge epithelium, exhibited a diminished amount of reaction product for both prostaglandins on day 13 as compared to the underlying mesenchyme. After formation of a midline epithelial seam between homologous palatal processes on day 14 of gestation, medial edge, oral, and nasal epithelium exhibited light staining for PGE or PGF2 alpha. Palate mesenchymal cells subjacent to the midline seam exhibited a diminished amount of reaction product for both PGE and PGF2 alpha as compared to day 13 of gestation. Overall, the results show local and temporal changes in the distribution of prostaglandins in the developing murine palate.

These were evaluated by: (1) a combined immunohistochemical-microphotometric procedure (IH) and (2) conventional radiometric assays. Human gingival fibroblasts were in the sixth passage, grown and maintained in Dulbecco minimal essential medium (DMEM) supplemented with 10 per cent horse serum. For chemical and hormonal stimuli, cells (2 x 10(4] were seeded on tissue-culture chamber/slides, and incubated with graded doses of either parathyroid hormone (PTH) or prostaglandin E2 (PGE2) for assessment of their adenosine-3',5'-monophosphate (cAMP) levels, and with indomethacin or colchicine for their effect on PGE levels. For mechanical stimuli, cells (1 x 10(6] were seeded on culture dishes with a flexible plastic membrane and stretched for 5, 30, 60 or 120 min by placing the membrane over a convex surface and weighting the dish cover. After freeze drying, cells were stained by an immunoperoxidase technique for either cAMP or PGE, using monoclonal antibodies. The staining intensity of fibroblasts was determined at 600 nm wavelength. Per cent light absorbance of 15 cells in each slide was measured and the results tested by analysis of variance. The gingival fibroblasts responded to the drugs and hormones in a dose- and time-related fashion. Stretching significantly increased their synthesis of PGE with concomitant increase in cAMP. The IH results were compared with the radiometric assays to confirm the validity of this technique; both assays were valid for describing the quantitative responses of these cells to the stimuli. In particular, the IH method could localize those intracellular sites which demonstrated chances in relative cAMP and PGE concentrations in response to hormonal stimuli.

The objective of this study was to examine the effect of smokeless tobacco (ST) on the development of the CD-1 mouse fetus. ST was administered continuously via Alzet osmotic minipumps during the critical gestational days 7-14 and 6-13. Two ST dosages were administered, 3.2 mg/ml (Dosage I) and 6.4 mg/ml (Dosage II), which yielded plasma nicotine levels within the range comparable to those of an average ST user or smoker (36.0 ng/ml). Plasma nicotine levels were maintained in the range of 29.4 +/- 4.8 ng/ml to 44.3 +/- 16.0 ng/ml for the Dosage I group of dams, and in the range of 34.6 +/- 10.9 ng/ml of 75.5 +/- 19.9 ng/ml for the Dosage II group of dams. The main effect on the fetus was weight reduction, with Dosage I producing a tendency toward weight reduction (p = .08). Dosage II produced a significant 8.6% weight reduction from normal (p less than .0001) and an increase in fetal deaths (p less than .03). Dosage I produced an increase in the incidence of hemorrhages and supernumerary ribs, and a significant delay (p less than .05) in ossification of the supraoccipital bone, the sacrococcygeal vertebrae, and the bones of the forefoot and hindfoot. There were no significant differences between placental weights. Weights of dams were significantly reduced only at the higher ST exposure levels. We conclude that at plasma nicotine levels comparable to those of an average ST user, ST produces weight reduction, delayed ossification, and increase in hemorrhages and fetolethality in the CD-1 mouse fetus.

The effect of verapamil on myocardial contractility, heart adenosine 3', 5'-monophosphate (cyclic AMP) and cardiac phosphorylase a activity was studied in isolated perfused rat hearts. In a concentration of 0.025 mug/ml, verapamil decreased force of contraction 50% and caused a significant fall in heart cyclic AMP within 4 to 5 min after perfusion with the drug was begun. When perfusion of the heart with medium containing verapamil was continued for 60 min, contractile force gradually returned to control. at the end of 60 min of perfusion with verapamil, the myocardial concentration of cyclic AMP was not different from that measured in hearts perfused with contro medium for a similar time period. Isoproterenol, given at the point of maximal contractile depression induced by verapamil, restored normal force of contraction and raised cardiac cyclic AMP to the same level as that observed when the catecholamine was given to untreated hearts; When methoxamine was administered to hearts depressed by verapamil, contractility returned to normal, but cyclic AMP content remained below control values.

Muscle cells dissociated from 5-day embryonic chicken hearts showed dose-dependent increases in both chronotropic rates of contraction and cyclic AMP (cAMP) levels in response to epinephrine (EPI), an effect that could be blocked by beta-adrenergic antagonists. However, 2- to 2.5-day embryonic chicken myocardial cells, although similar to 5-day heart cells with respect to the organization of myofibrils, failed to respond to EPI either by increased rates of contraction or by elevated levels of intracellular cAMP. Development of beta-adrenergic sensitivity in 2- to 2.5-day cells did not occur even after several days of growth in culture. However, addition of an extract prepared from 11-day chicken embryos to 2- to 2.5-day muscle cell cultures at any point during in vitro growth resulted in the development of sensitivity to EPI as measured by increases in both the beating frequency and cAMP levels after a 48-hr incubation in embryo extract (EE). The basal level of cAMP in cells unresponsive to EPI is 5 times that in EPI-sensitive muscle cells from older hearts (5 days). The high basal level of cAMP in these cells is reduced to a level characteristic of cells from older hearts when treated with the EE. Once sensitivity was acquired, it was retained as a stable trait of the muscle cells in culture. Furthermore, EE-treated 2- to 2.5-day cells showed less reduction of positive chronotropy in response to multiple doses of EPI than did cells prepared from 5-day hearts. Fractionation of EE on Sephadex G-200 showed that the activity was present in the large-molecule fractions. It is concluded that the development of beta-adrenergic sensitivity can be induced in unresponsive heart cells cultured from 2- to 2.5-day embryos by a factor(s) in the EE that is not adsorbed on Sephadex G-200.

The effects of verapamil on myocardial isometric force on contraction, cardiac adenosine 3,'5'-monophosphate (cyclic AMP) and heart phosphorylase alpha activity were studied in the isolated perfused rat heart. When hearts were perfused with verapamil (5.98 times 10- minus 8 M), force of contraction was reduced approximately 50% within 4 to 5 minutes; at this point, the concentration of cyclic AMP was significantly lower than control but phosphorylase alpha activity was unchanged. In hearts perfused continuously for 60 minutes with verapamil, force of contraction and cyclic AMP levels returned to normal within 20 minutes after administration of verapamil was begun. Isoproterenol (0.355 nmol/min) reversed the depressant effect of verapamil on cardiac contractility and restored heart cyclic AMP levels to normal. Methoxamine (35.5 nmol/min) given to verapamil-depressed hearts, caused contractile force to return to normal, but cardiac cyclic AMP levels remained low. Mephentermine (23.0 nmol/min) had no effect on cardiac contraction, cyclic AMP or phosphorylase alpha activity in hearts depressed by verapamil. It was concluded that with the concentration of verapamil used in these experiments, the drug caused a transient decrease in force of contraction and myocardial cyclic AMP. Both the depression in myocardial contractility and in cardiac cyclic AMP caused by verapamil were reversed promptly by isoproterenol, whereas methoxamine overcame acutely only the negative inotropic effect of verapamil. Mephentermine had no effect on hearts depressed by verapamil.