Paper review

The Obestatin/Ghrelin System as a Novel Regulatory Mechanism of Iris Muscle Contraction
A. Rocha-Sousa, F. Falcão-Reis, A. F. Leite-Moreira
Current Eye Research, Volume 33, Issue 1 January 2008 , pages 73 - 79
(link)

Background:

Obestatin is a 23aa amidated peptide derived from preproghrelin, a product of the ghrelin gene. It is known to activate a rodopsin type G-coupled receptor (GPR-39), with binding sites in the GI tract and both the pituitary and hypothalamus. It tends to be co-localized with acetylcholine in the myenteric plexus, which provides innervation to the smooth muscles of the GI tract. Therefore it may be a regulator of cholinergic muscle contraction.

Ghrelin is a 28aa peptide also produced from the ghrelin gene. It is found in the GI tract, pituitary and hypothalamus, and ghrelin mRNA has been found in the iris posterior epithelium and non-pigmented ciliary epithelial cells in rat's eyes. It has been suggested that ghrelin acts on smooth muscles and it has been implicated in relaxation of the iris sphincter and dilator muscles.

There is evidence that obestatin and ghrelin may have opposite effects in metabolic and GI function. For example, ghrelin stimulates feeding in some animals and increases during fasting, while obestatin injections suppress weight gain and delay gastric emptying.

Iris sphincter muscle contraction is mediated by acetylcholine release and can be modulated by the action of several peptides and hormones. Iris dilator muscle contraction is controlled by the adrenergic system whose modulation is less well known.

This study aims to describe the effects of obestatin and ghrelin on contraction and relaxation of iris sphincter and dilator muscles.

Methods:

Iris sphincter (n=60) and dilator (n=48) muscles were isolated from New Zealand white rabbits (Oryctolagus cuniculus) and immersed in a buffer solution. The ends of each muscle were tied with silk thread and mounted horizontally, one end connected to an electromagnetic length-tension transducer. Muscles were allowed to extend until length remained constant and muscle tension stabilized.

1-5 human ghrelin (a 5aa fragment acting as ghrelin agonist) and human obestatin were used.

First iris sphincter and dilator muscle contractions were elicited by addition of either carbachol (acetylcholine agonist, 10^-6M) or epinephrine (10^-4M), respectively. Then the bath was washed out and after the muscles were stabilized for 5 minutes a second contraction was induced in the additional presence of either obestatin (10^-5M) or 1-5 ghrelin (10^-5M). Muscle tensions generated in the second versus the first contraction of iris sphincter and dilator muscles were compared to the vehicle (control, no obestatin/ghrelin added). This process was repeated using muscle contraction elicited by electric field stimulation (EFS) of 10V, 100Hz during 1ms instead of carbachol or epinephrine.

Finally concentration-response curves were generated by recording muscle tension of iris sphincter and dilator muscles with increasing concentrations of 1-5 ghrelin and obestatin (between 10^-9 and 10^-5M), expressing effects on muscle tension as percent change from initial value.

Results:


Figure 1: Representative example of iris sphincter muscle active tension in two consecutive carbachol-elicited contractions in the absence or presence of: (1) the vehicle (Panel A); (2) obestatin (10-5 M; Panel B); or (3) ghrelin (10-5 M; Panel C). (Rocha-Sousa et al., 2008)

Effects of obestatin and ghrelin on active tension produced by carbachol-induced contractions of iris sphincter muscle (Figures 1 and 2)

• No significant difference in 1st contraction among 3 groups
  
• Significant (p<0.05) increase (19.0 +/- 10.2%) in presence of obestatin
  
• Decrease in vehicle-only (11.5 +/- 5.5%) but insignificant (p=0.06)

Figure 2: Iris sphincter muscle tension (upper panel) of two consecutive carbachol-induced (10-6 M) contractions in the absence (first contraction, left column) or presence (second contraction, right column) of the vehicle alone (n= 8), obestatin (10-5 M; n= 10) or ghrelin (10-5 M; n= 7). The lower panel represents the percent tension variation from the first to the second contraction in the same experimental conditions (p < 0.05: *second versus first contraction). (Rocha-Sousa et al., 2008)

Effects of obestatin and ghrelin on active tension produced by epinephrine-induced contractions of iris dilator muscle (Figure 3)

• No significant difference in 1st contraction among 3 groups
  
• Decrease (10.7 +/- 2.7%, p<0.05) in vehicle-alone
  
• No significant change with addition of obestatin or ghrelin

Figure 3: Iris dilator muscle tension (upper panel) of two consecutive epinephrine-induced (10-4 M) contractions in the absence (first contraction, left column) or presence (second contraction, right column) of the vehicle alone (n=8), obestatin (10-5 M; n=8) or ghrelin (10-5 M; n=8). The lower panel represents the percent tension variation from the first to the second contraction in the same experimental conditions (p < 0.05: *second versus first contraction). (Rocha-Sousa et al., 2008)

Effects of obestatin and ghrelin on active tension produced by electric field stimulation-induced contractions of iris sphincter muscle (Figure 4)

• No significant difference in 1st contraction among 3 groups
  
• No significant differences between 1st and 2nd contractions in vehicle-only and obestatin-added groups
  
• Decrease (0.86 +/- 0.06 mN, p<0.05) with addition of ghrelin

Figure 4: Iris sphincter muscle tension (upper panel) of two consecutive EFS-elicited contractions in the absence (first contraction, left column) or presence (second contractions, right column) of the vehicle alone (n = 8), obestatin (10-5 M; n = 8) or ghrelin (10-5 M; n = 8). The lower panel represents the percent tension variation from the first to the second contraction in the same experimental conditions (p < 0.05: **second versus first contraction). (Rocha-Sousa et al., 2008)

Effects of increasing obestatin/ghrelin concentrations on basal muscle tension

• No significant differences in resting tensions at baseline
  
• Only ghrelin significantly altered sphincter muscle tension, decreasing 21.7 +/- 3.7% at 10^-5M (p<0.05 vs control)
  
• Only obestatin significantly altered dilator muscle tension, decreasing 14.1 +/- 5.0% at 10^-5M (p<0.05 vs control)

Figure 5: Concentration-response curves of obestatin (10-9–10-5M) and ghrelin (10-9–10-5M) on the basal tension of the iris sphincter (upper panel) and dilator (lower panel) muscles (p < 0.05: *versus control). (Rocha-Sousa et al., 2008)

Summary of results:

In iris sphincter muscles:

• Obestatin potentiates cholinergic contraction elicited by carbachol
  
• Ghrelin decreases basal tension in a concentration-dependent manner
  
• Ghrelin decreases tension developed in EFS-elicited contraction

In iris dilator muscles:

• Obestatin decreases basal tension in a concentration-dependent manner

Critique:

The figures were simple and clearly demonstrated the differences in muscle tension generated in all treatments of iris muscle contractions, including their significance.

The experiment provides convincing evidence that obestatin and ghrelin may have opposite effects on muscle contraction and relaxation in the iris sphincter muscles. Obestatin appears to potentiate cholinergic contraction in a similar mechanism to that proposed for the GI tract, although it had an opposite relaxing effect on the iris dilator muscles. The evidence suggests that obestatin is moreso involved in pupil constriction while ghrelin is involved in dilation, but ghrelin failed to show any direct action on the iris dilator muscles.

A possible reason for a lack of response to ghrelin in iris dilator muscles is the fact that a 5aa fragment of human ghrelin is used in the experiment. Perhaps a fragment so small or even a complete human peptide would be insufficient to stimulate the applicable Oryctolagus receptor so a possible effect in vivo cannot be ruled out. Since both obestatin and 1-5 ghrelin were obtained from human sources it is possible that both peptides may have different affinities to the foreign receptors so actual increases or decreases in muscle tension in vivo cannot be quantified from this experiment. However, the fact remains that some effects were seen and this is strong evidence for involvement of both obestatin and ghrelin in mediation of iris sphincter and dilator muscles.

References:
Rocha-Sousa A., Falcão-Reis F., Leite-Moreira A.F. 2008 The Obestatin/Ghrelin System as a Novel Regulatory Mechanism of Iris Muscle Contraction. Current Eye Research, 33(1):73-79

The Iris: Introduction

Figure 1: Photo of a human eye showing the iris and pupil (http://en.wikipedia.org/wiki/Image:Bluishgrayeye.JPG)

The Iris

The iris is an extension of the choroid partially covering the lens of the eye leaving a central circular aperture called the pupil (Junqueira and Carneiro, 2005).


Figure 2: Diagram of the eye showing position of iris (Click for bigger) (Junqueira and Carneiro, 2005)


Figure 3: Section of iris, showing inner layer (adjacent to anterior chamber), outer layer (adjacent to posterior chamber) and the dilator and constrictor muscles of the iris (PT stain, click for bigger) (Junqueira and Carneiro, 2005)

The layers of the pupil are as follows (from Junqueira and Carneiro, 2005):

• Anterior surface: rough and irregular, a discontinuous layer of pigment cells and fibroblasts
  
  
• A layer of poorly vascularized connective tissue with few fibers, and many fibroblasts and melanocytes
  
  
• A layer of loose connective tissue rich in blood vessels
  
  
• Posterior surface: smooth, covered by 2 layers of epithelium which also cover the ciliary body and its processes
  
  
• Inner epithelium: heavily pigmented with melanin granules, in contact with posterior chamber. The outermost cells have radial tongue-like extensions and contain overlapping myofilaments
  

The abundance of melanocytes and intensity of pigmentation in the iris prevents stray light rays from entering the eye except through the pupil, which would interfere with image formation (Junqueira and Carneiro, 2005).

The melanocytes found within the connective tissue of the iris are responsible for eye colour: if only a few pigment cells are present, the light reflecting from the black pigment epithelium in the posterior surface will be blue (Junqueira and Carneiro, 2005). As the cell number increases, the iris colour becomes greenish-blue, gray, and finally brown (Junqueira and Carneiro, 2005). Albinos, having very little pigment, have a pink eye colour due to light reflecting from iris blood vessels (Junqueira and Carneiro, 2005).

Myofilaments of the inner iris epithelium form the dilator pupillae muscle, which runs radially from the pupil to the iris margin (Junqueira and Carneiro, 2005). Smooth muscle bundles concentric with the pupillary margin form the sphincter pupillae muscle imbedded in the connective tissue of the iris (Junqueira and Carneiro, 2005). The dilator and sphincter muscles work in concert to increase and decrease the pupil diameter, respectively (Junqueira and Carneiro, 2005). Innervation of the dilator muscle is sympathetic, and the sphincter muscle, parasympathetic (Junqueira and Carneiro, 2005).

The iris, along with the ciliary region and the retinal pigment epithelium, is formed from the neurectoderm during embryonic eye development (Akagi and Takahashi, 2005).


Figure 4: Development of the eye. (A) Evagination of the primary optic vesicle. (B) Invagination of the surface epithelium and formation of the optic cup by invagination at the embryonic fissure. (C) Detachment of the lens from the surface epithelium. (D) The ciliary region and iris are formed from neuroectoderm, and the lens from the posterior cells of the lens vesicle. (E) The completed eye. IPE, iris pigmented epithelium; RPE, retinal pigment epithelium; CB, ciliary body. (Akagi and Takahashi, 2005)

Iris-derived cells have been induced to express rod photoreceptor genes and to take the form of retinal rod cells, suggesting a possibility of retinal repair grafts from iris tissue (Akagi and Takahashi, 2005).

Iris melanoma

Iris melanoma (cancer of pigment cells or melanocytes) is the most common malignancy of the iris, though the iris is the least common site of primary uveal (involving the iris, ciliary body or choroid) melanoma (Henderson, 2008). It also has a better prognosis than other uveal melanomas (Henderson, 2008).

Victims of iris melanoma have an average age in the mid-late 40's, and the disease tends to occur most often in persons with fair complexions and blue to gray iris colour (Henderson, 2008). Interestingly, 80% of iris melanomas arise in the inferior half of the iris (Henderson, 2008).

There are two main types of iris melanomas: a) circumscribed, consisting of a yellow/tan/brown pigmented spot, and b) diffuse, presenting as a unilateral darkening of the iris (Henderson, 2008). Circumscribed iris melanomas are the easiest to detect (Henderson, 2008).


Figure 5: Circumscribed iris melanoma at approximately 10 o’clock position. The brown tumor arises at the root of the iris and protrudes into the anterior chamber (Henderson, 2008)


Figure 6: Diffuse iris melanoma. The iris developed its brown and gray color during 1 year. The opposite eye was blue. (Henderson, 2008)

The tumour usually consists mostly of uniform spindle melanoma cells, and some contain epithelial cells (Henderson, 2008). More than 50% of iris melanomas are reported as "spindle cell" melanomas, which have the best prognosis (Henderson, 2008).


Figure 7: A) Circumscribed iris melanoma fills the anterior chamber. The tumor is cellular and bulky (X2 Mag.). B) A majority of the tumor was composed of relatively uniform spindle melanoma cells, most of which contain visible melanin (X40). C) A minority of the tumor contained epithelioid cells, characterized by their larger size, greater pleomorphism, eosinophilic cytoplasm, and larger nuclei with prominent nucleoli (X40). All taken using hematoxylin-eosin staining (Click for bigger) (Henderson, 2008)

A limited number of treatments for growing iris melanomas are available. Most discrete lesions are excised surgically, preserving vision (Henderson, 2008). Tumours involving the angle of the anterior chamber require partial resection of the ciliary body, which involves an increased risk of ocular morbidity (loss of an eye) (Henderson, 2008). The procedure is difficult because a small specimen must be properly oriented and uveal tissue is easily crushed by handling (Henderson, 2008). Radiotherapy is not often used to manage resectable iris melanomas because of an increased risk of vision complications (Henderson, 2008). Particularly large or diffuse tumours (involving more than 50% of the iris) usually require enucleation (removal of the eye) (Henderson, 2008). For patients for which this is undesirable (for example, one-eyed persons) there are possible alternatives to enucleation including the use of specially designed radioplaques for treatment of iris melanoma (Henderson, 2008).

References:

Akagi, T. and Takahashi, M. 2005 Photoreceptors Derived from Adult Iris Tissue: Prospects for Retinal Transplantation. Seminars in Ophthalmology 20(1);11-15

Henderson E., Margo C.E. 2008. Iris Melanoma. Arch Pathol Lab Med. 132:268-272

Junqueira L.C., Carneiro J. 2005. Basic Histology, Eleventh Edition. The McGraw-Hill Companies, Inc. United States of America

First post

Hat trick!